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Os09g0422500

2014-06-10T06:31:54Z

Haoyajing cathy:

The Os09g0422500 gene can be called by another gene symbols like BC6 and OsCESA9 which similar to cellulose synthase .The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle phenotype in the culm and plays an important role in cell wall biosynthesis and plant growth.

==Annotated Information==
===Introduction===
A genomic fragment containing the mutant Bc6 gene (9374 bp), including 4.8 kbp of upstream sequence and 492 bp downstream, was digested with HindIII and subcloned into the binary vector pBIGRZ to yield the mutant construct, gBc6/pBIGRZ. The wild-type BC6 gene construct, gBC6/pBIGRZ, was generated by changing the mutated nucleotide at position 7112 from G to wild-type A by PCR mutagenesis with primers, PM-F1 (5′-GCAGTTCCCGCAGAGGTTCGACGGC-3′) and PM-R1 (5′-ATCGACGTCCACGACCGATACGCC-3′). These constructs were introduced into the wild-type plant, T65, by an Agrobacterium (Rhizobium radiobacter)-mediated method using strain EHA101. The T2 generation of transgenic plant was used for cell wall analysis.
[[File: haoyajing.jpg|right|thumb|250px|'''Figure 1.''' ''rice from Brittle phenotype lines VS. Normal lines ]]

1.Promoter

PromoterBC6:β-glucuronidase (pBC6:GUS) activity was assayed according to the method of Kosugi et al.The 4.8 kbp promoter region of BC6 was amplified by PCR using specific primers, gBc6-F1 and pBC6-R1 (5′-GAGGATCCATGGCCGCGCAACAACGGCCGG-3′), and fused with the GUS gene in the pBIGRZ vector, yielding pBC6:GUS. The nucleotide sequence of the construct was confirmed before transformation into the wild-type plant, T65, as described above. Culms, leaves, nodes, and seedlings of the transgenic plants harbouring the pBC6:GUS gene were cut into pieces, fixed in 5% (w/v) agar, and hand-sectioned. Sections from the transgenic plants were stained with a solution containing 0.5 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 50 mM phosphate buffer (pH 7.4) at 37 °C for 24 h, and observed under a microscope (Eclipse E400).

2.Quantitative analysis of BC1 and BC6 mRNAs

Relative amounts of BC1, BC3, BC6, OsCesA4, and OsCesA7 mRNA were estimated by quantitative RT-PCR. Single-stranded cDNA was synthesized from total RNA of the tissues or organs using oligo(dT)12–18 primer. The following specific primers were designed using the Primer3 program (http://frodo.wi.mit.edu/): for BC1 (Os03g0416200), BC1-RTP-F1 (5′-CGCATGAACTACACCCAGTG-3′) and BC1-RTP-R1 (5′-TCCATGAGCAGGTCGTTGTA-3′); for BC3 (Os02g0738900), BC3-RTP-F1 (5′-GGCCGAAACGATGAGATTTA-3′) and BC3-RTP-R1 (5′- AACATCAGCAGCTTGCATTG-3′); for BC6 (Os09g0422500), BC6-RTP-F1 (5′-TTAGCACGTTTGCGAGTTTG-3′) and BC6-RTP-R1 (5′-GAACTCGTCGTCCTCGTCTC-3′); for OsCesA4 (Os01g0750300), OsCesA4-RTP-F1 (5′-CTAATGCGACGAAGACGATG-3′) and OsCesA4-RTP-R1 (5′-GATTTAACGGTGCCCTCTCA-3′); for OsCesA7 (Os10g0467800), OsCesA7-RTP-F1 (5′-TCCATCTTCTCCCTCGTCTG-3′) and OsCesA7-RTP-R1 (5′-GAATCATCCATCCGGTCATC-3′); and for ACTIN1 (Os03g0718100), ACT1-RTP-F1 (5′-TTCCTACATCGCCCTGGACT-3′) and ACT1-RTP-R1 (5′-AGCCTTGGCAATCCACATCT-3′). The PCR was performed with a SYBR Premix Ex Taq kit under the following conditions: 10 s denaturing at 95 °C, 30 s annealing at 60 °C, and 20 s amplification at 72 C, 40 cycles. The PCR products were detected with Opticon 2 , and the mRNA amounts relative to ACTIN1 mRNA were calculated.

===Function===
1;This study characterizes a brittle culm (bc88) mutant of rice (Oryza sativa L.) obtained by ethylene methylsulfonate (EMS)-induced mutagenesis of Wuyunjing 7. The bc88 mutant exhibits a diversity of pleiotropic phenotypes, including brittle culm at the whole-plant growth stages, withered leaf tips at the seedling stage, and 18-d delay in heading date at the mature stage. Genetic analysis indicates that the bc88 mutant is controlled by a single recessive nuclear gene. The mutated bc88 gene isolated by map-based cloning contains only one point mutation in the 5th exon relative to its wild-type BC88 (LOC_Os09g25490 and Os09g0422500), leading to an amino acid change from P to L in bc88 plants. Alignment of the putative protein sequence with its homologs indicates that the mutation is located in the conserved region of the sequence. Detection of the transcription level of BC88 in rice plants shows that the expression level of BC88 is higher in spikes and culms than in leaves, roots, and leaf sheaths. These contribute to understanding of the molecular mechanism of cellulose synthesis. The target gene BC88 can be a useful tool in molecular marker-assisted selection for rice culm trait breeding.

2;Rice is a model organism in poaceae plants to study cell wall biosynthesis. In this study, a mutant S1-60 isolated from an EMS mutagenized japonica cultivar Nipponbare, is characterized by brittle culms that can be easily broken by bending. The reduction in mechanical strength was due to defect in thickening of the sclerenchyma cell wall. The amount of cellulose in S1-60 culms was reduced to 44.7% of that of wild-type plants. Besides, the mutant also exhibited pleiotropic phenotypes, such as dwarfism and partial sterility. Genetic analysis and map-based cloning showed that all the phenotype of S1-60 mutant was caused by a recessive point mutation in the OsCESA9 gene, which encodes the cellulose synthase A subunit 9. This yet uncharacterized missense mutation changed the highly conserved G905 to D at the beginning of the fifth transmembrane domain. The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle phenotype in the culm. These results indicate that OsCESA9 plays an important role in cell wall biosynthesis and plant growth.

3;According to the MSU Rice Genome Annotation Release 7 (http://rice.plantbiology.msu.edu), there are 13 predicted open reading frames (ORFs) within the 118 kb fine mapping interval , including 7 ORFs with known biochemical functions, 5 ORFs encoding expressed hypothetical protein and 1 transposon . Among them, ORF3 (TIGR ID: LOC_Os09g25490) encoding cellulose synthase A catalytic subunit 9 (OsCESA9) was considered as the priority candidate gene, given that the amount of cellulose was reduced dramatically in S1-60 culm. Therefore, we sequenced and compared the mutant and wild type alleles of the OsCESA9 gene, which has 4643 bp in length and 11 exons and 10 introns. One base pair substitution was found in the last exon, changing GGC to GAC and the encoded amino acid from glycine to aspartic acid at the 905th position .The CESA9 protein has 1056 amino acids in length and eight putative transmembrane domains (TMDs), with two near the amino terminus and six clustered near the carboxyl terminus . There is a RING-type zinc finger in the N-terminal region, which might mediate the interaction between CESA9 and other CESA subunits. A large central domain containing two highly conserved motifs DXD and Q/RXXRW is required for the catalytic activity of the enzyme. The missense mutation in S1-60 occurs at the beginning of the fifth TMD, which might affect the plasma membrane localization of CESA9.

4;The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.

===Expression===
We examined the expression level of OsCESA9 in various rice organs by both semi-quantitative and quantitative RT-PCR. The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle culm phenotype in the S1-60 mutant. The OsCESA9 gene is also expressed in panicle at mature stage. However, the expression level of OsCESA9 was relatively low at seedling stage, no matter in root, leaf blade or leaf sheath. This result showed the OsCESA9 gene mainly participates in the synthesis of secondary cell wall in mechanical tissue at late development stage.

The pattern of expression of BC6 was assessed by analysing T65 plants transformed with pBC6:GUS, a binary vector in which the 4.8 kbp region upstream of the BC6 gene was fused with the GUS reporter gene. BC6 promoter activity was detected in leaves, culms, and nodes, with relatively strong expression in culm vascular bundles 2 weeks after heading.Promoter activity was also observed in young tissues such as developing leaves. Although Bc6 mutation appeared to reduce cell wall thickness in sclerenchymal cells , promoter activity was not detected in developed sclerenchymal cells .It is possible that the reporter gene activity did not completely mirror the expression of the BC6 gene product, OsCesA9 protein. These patterns of BC6 promoter-driven gene expression were similar to the expression pattern of BC1 demonstrated by in situ hybridization .

In Arabidopsis, the COBL4 gene is co-expressed with the secondary cell wall-specific CesA genes and is presumed to play a role in the cellulose synthesis of secondary cell walls, although the precise molecular functions of COBRA-like proteins remain unclear .To examine the relationship between the secondary cell wall-specific CesA and COBRA-like proteins in rice, BC6 and BC1 mRNAs were quantitated in several tissues. Consistent with the results of the pBC6:GUS analysis, relatively high levels of BC6 mRNA were detected in culms and nodes.The level of Bc6 mRNA in roots was relatively low. Indeed, the brittle phenotype in roots was not clear compared with those in culms and leaves.Despite the apparent brittle phenotype in the leaves of Bc6 mutants, the level was low in both leaf blades and sheaths. Importantly, both BC6 and BC1 were highly expressed in culms, nodes, and flowers .These results indicated that BC6 and BC1 are co-expressed during development of secondary cell walls. On the other hand, the expression of BC6 was not related to that of BC3, suggesting that BC6 and BC3 are differently regulated.

The effect of the mutation on the expression of Bc6 was also examined in developing leaf blades. Bc6 mutants showed accumulation of BC6 mRNA comparable with T65.Furthermore, Bc6 mutation barely influenced the mRNA levels of other CesA genes, OsCesA4 and OsCesA7, which are expected to participate in cellulose synthesis in secondary cell walls, together with BC6 (OsCesA9).

===Evolution===
Please input evolution information here.

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==Labs working on this gene==
1. College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao 266109, China

2. National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

3. Shandong Rice Research Institute, Jinan 250100, China

4. College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China

5. Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan

6. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China;

7. State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China

==References==
1.A missense mutation in the transmembrane domain of CESA9 affects cell wall biosynthesis and plant growth in rice. Wang D, et al. Plant Sci, 2012 Nov. PMID 23017906

2.Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls. Kotake T, et al. J Exp Bot, 2011 Mar. PMID 21209026, Free PMC Article

3.The Rice Annotation Project Database (RAP-DB): 2008 update. Rice Annotation Project, et al. Nucleic Acids Res, 2008 Jan. PMID 18089549, Free PMC Article

4.Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana. Rice Annotation Project, et al. Genome Res, 2007 Feb. PMID 17210932, Free PMC Article

5.The Rice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp. japonica genome information. Ohyanagi H, et al. Nucleic Acids Res, 2006 Jan 1. PMID 16381971, Free PMC Article
==Structured Information==
{{JaponicaGene|
GeneName = Os09g0422500|
Description = Similar to Cellulose synthase (Fragment)|
Version = NM_001069742.1 GI:115479226 GeneID:4347093|
Length = 4574 bp|
Definition = Oryza sativa Japonica Group Os09g0422500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 9|Chromosome 9]]|
AP = Chromosome 9:15935031..15939604|
CDS = 15935124..15935394,15935500..15935735,15935831..15935927,15936019..15936145,15936232..15936844 ,15936934..15937197,15937280..15937492,15937615..15937822,15937914..15938110 ,15938192..15938545,15938649..15939236|
GCID = <gbrowseImage1>
name=NC_008402:15935031..15939604
source=RiceChromosome09
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008402:15935031..15939604
source=RiceChromosome09
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggaggcgagcgccgggctggtggccgggtcgcacaaccggaacgagctggtgctgatccgggggcacgaggagcccaagccgctgcgggcgctgagcgggcaggtgtgcgagatatgcggcgacgaggtcggccgcaccgtcgacggcgacctcttcgtcgcctgcaacgagtgcggcttcccggtgtgccgcccctgctacgagtacgagcgccgcgagggcacccagaactgcccccagtgcaagacccgctacaagcgcctcaaggggagcccgagggtgcccggggacgaggacgaggaggacattgacgacctggagcacgagttcaacatcgacgacgagaagcagaagcagctgcagcaggatcaggatggcatgcagaacagccacatcaccgaggcgatgctgcacggcaagatgagctacgggaggggccccgacgacggcgacggcaacagcaccccgctcccgccgatcatcaccggcgctcgctccgtcccggtgagcggggagttcccgatatcgaacagccatggccatggcgagttctcctcttccctgcacaagcgcatccacccctacccggtgtctgagccagggagtgcaaagtgggacgagaagaaagaggtgagctggaaggagaggatggacgactggaaatccaagcagggcatcgtcgccggcggcgcccccgatcccgacgactacgacgccgacgtcccactgaacgacgaggcgaggcagccgctgtcgaggaaggtgtcgatcgcgtcgagcaaggtgaacccgtaccggatggtgatcatcctccgtctcgtggtgctcggcttcttcctccggtaccgcatcctccacccggtgcccgacgccatcccgctgtggctcacctccatcatctgcgagatctggttcgccgtgtcgtggatcctcgaccagttccccaagtggtacccgatcgacagggagacctacctcgaccgcctctccctccgctacgagcgcgagggggagccgtcgctgctgtcggcggtggacctgttcgtcagcacggtggatccgctcaaggagccgccgctggtcacggccaacaccgtgctgtccatcctcgccgtcgactaccccgtcgacaaggtgtcctgctacgtctccgacgacggcgcgtccatgctcacgttcgagtcgctgtcggagacggcggagttcgcccgcaagtgggtgccattctgcaagaagttcagcatcgagccccgcgccccggagttctacttctcccagaaggtcgactacctcaaggacaaggtccatcccaacttcgtccaggagcgccgcgccatgaagagagagtacgaggagttcaaggtgaggatcaacgcgctggtggcgaaggcgcagaaggtgccggcggaagggtggatcatgaaggacgggacgccatggccggggaacaacacccgcgaccacccgggcatgatccaggtgttcctcggccacagcggcggccacgacaccgagggcaacgagctcccccgcctcgtctacgtctcccgtgagaagcgccccggcttccagcaccacaagaaggccggcgccatgaacgccctcattcgtgtgtcggccgtgctgacgaacgcgccgttcatgctcaacttggattgcgatcactacatcaacaacagcaaggccatcagggaggcgatgtgcttcctcatggatccgcaggtcggacggaaggtttgctacgtgcagttcccgcagaggttcgacggcatcgacgtccacgaccgatacgccaaccgcaacaccgtcttcttcgacatcaacatgaaggggcttgatgggatccagggcccggtgtacgtcgggacagggtgcgtgttcaggcggcaggcgctgtacggatacaacccacccaagggacccaagaggcccaagatggtgacctgcgactgctgcccttgcttcgggaggaagaagcggaagcacggcaaggacggcctcccggaggccgtcgccgccgacggcgggatggacagcgacaaggagatgctcatgtcgcagatgaacttcgagaagcggttcgggcagtcggcggcgttcgtgacgtcgacgctgatggaggaaggcggcgtcccgccgtcgtccagccccgccgcgctcctcaaggaggccatccatgtcatcagctgcggctacgaggacaagaccgactggggtctcgagctggggtggatctacgggtcgatcacggaggacatcctaacggggttcaagatgcactgccgcgggtggaggtcggtgtactgcatgccgaagagggcggcgttcaaggggtcagcgccgatcaacctatctgaccgtctcaaccaggtgctccggtgggcgctcggctccgtcgagatcttcttcagccggcacagcccgctcctctacggctacaagaacggcaacctcaagtggctcgagcgcttctcctacatcaacaccaccatctaccccttcacttctctccccctcctcgcctactgcaccctacccgccgtctgcctcctcaccggcaagttcatcatgcctccgattagcacgtttgcgagtttgttcttcatcgcgctcttcatctccatcttcgcgacgggcatcctggagatgaggtggagcggggtgagcatcgaggagtggtggaggaacgagcagttctgggtcatcggcggcgtgtcggcgcacctgttcgcggtggtgcagggcctgctcaaggtgctggccgggatcgacaccaacttcaccgtcacgtccaaggccaccggagacgaggacgacgagttcgcggagctctacgccttcaagtggaccaccctcctcatcccgcccaccacgctgctcatcctcaacatcatcggcgtcgtcgccggcgtctccgacgccatcaacaacggctccgaggcgtggggcccgctcttcgggaagctcttcttcgccttctgggtcatcgtccacctctaccccttcctcaaggggctcatggggaggcagaaccggacgcccaccattgttgtcatctggtccgtgctgctcgcctccatcttttccttgctctgggtcaggattgatcccttcaccatcaaggccaggggccctgacgtcaggcagtgcggcatcaactgctga</cdnaseq>|
AA = <aaseq>MEASAGLVAGSHNRNELVLIRGHEEPKPLRALSGQVCEICGDEV GRTVDGDLFVACNECGFPVCRPCYEYERREGTQNCPQCKTRYKRLKGSPRVPGDEDEE DIDDLEHEFNIDDEKQKQLQQDQDGMQNSHITEAMLHGKMSYGRGPDDGDGNSTPLPP IITGARSVPVSGEFPISNSHGHGEFSSSLHKRIHPYPVSEPGSAKWDEKKEVSWKERM DDWKSKQGIVAGGAPDPDDYDADVPLNDEARQPLSRKVSIASSKVNPYRMVIILRLVV LGFFLRYRILHPVPDAIPLWLTSIICEIWFAVSWILDQFPKWYPIDRETYLDRLSLRY EREGEPSLLSAVDLFVSTVDPLKEPPLVTANTVLSILAVDYPVDKVSCYVSDDGASML TFESLSETAEFARKWVPFCKKFSIEPRAPEFYFSQKVDYLKDKVHPNFVQERRAMKRE YEEFKVRINALVAKAQKVPAEGWIMKDGTPWPGNNTRDHPGMIQVFLGHSGGHDTEGN ELPRLVYVSREKRPGFQHHKKAGAMNALIRVSAVLTNAPFMLNLDCDHYINNSKAIRE AMCFLMDPQVGRKVCYVQFPQRFDGIDVHDRYANRNTVFFDINMKGLDGIQGPVYVGT GCVFRRQALYGYNPPKGPKRPKMVTCDCCPCFGRKKRKHGKDGLPEAVAADGGMDSDK EMLMSQMNFEKRFGQSAAFVTSTLMEEGGVPPSSSPAALLKEAIHVISCGYEDKTDWG LELGWIYGSITEDILTGFKMHCRGWRSVYCMPKRAAFKGSAPINLSDRLNQVLRWALG SVEIFFSRHSPLLYGYKNGNLKWLERFSYINTTIYPFTSLPLLAYCTLPAVCLLTGKF IMPPISTFASLFFIALFISIFATGILEMRWSGVSIEEWWRNEQFWVIGGVSAHLFAVV QGLLKVLAGIDTNFTVTSKATGDEDDEFAELYAFKWTTLLIPPTTLLILNIIGVVAGV SDAINNGSEAWGPLFGKLFFAFWVIVHLYPFLKGLMGRQNRTPTIVVIWSVLLASIFS LLWVRIDPFTIKARGPDVRQCGINC</aaseq>|
DNA = <dnaseqindica>94..364#470..705#801..897#989..1115#1202..1814#1904..2167#2250..2462#2585..2792#2884..3080#3162..3515#3619..4206#agcgatcgatcgcccttcctcctcctcctctcctccttcctgcgtcgcctccctgatcagccgcagctcgttgccggccgttgttgcgcggccatggaggcgagcgccgggctggtggccgggtcgcacaaccggaacgagctggtgctgatccgggggcacgaggagcccaagccgctgcgggcgctgagcgggcaggtgtgcgagatatgcggcgacgaggtcggccgcaccgtcgacggcgacctcttcgtcgcctgcaacgagtgcggcttcccggtgtgccgcccctgctacgagtacgagcgccgcgagggcacccagaactgcccccagtgcaagacccgctacaagcgcctcaagggtaaaaaaacacactcacatcacgctacgcccttgataccgcgatcgcgaggtttccgttgattgatctctaatggcgatggtggtggtggtggttttgttacagggagcccgagggtgcccggggacgaggacgaggaggacattgacgacctggagcacgagttcaacatcgacgacgagaagcagaagcagctgcagcaggatcaggatggcatgcagaacagccacatcaccgaggcgatgctgcacggcaagatgagctacgggaggggccccgacgacggcgacggcaacagcaccccgctcccgccgatcatcaccggcgctcgctccgtcccggtacacacaacacacctcaccccactcacaccaaattctctccctcttctcacctcaacatgttcacgaaatgttctttgtaaaaaaaaattcaggtgagcggggagttcccgatatcgaacagccatggccatggcgagttctcctcttccctgcacaagcgcatccacccctacccggtgtctgagccaggtagtataacgaattcactacactaattaatcaatgttcttgatgaacacacattgatcatctcaatcatctaccgatgaattctgaccagggagtgcaaagtgggacgagaagaaagaggtgagctggaaggagaggatggacgactggaaatccaagcagggcatcgtcgccggcggcgcccccgatcccgacgactacgacgccgacgtcccactgtacgcaatcctcagctgagcagcttacactgatcatgcatgacaactagtatattgatcatgcctgaactctctgtggcttgcaggaacgacgaggcgaggcagccgctgtcgaggaaggtgtcgatcgcgtcgagcaaggtgaacccgtaccggatggtgatcatcctccgtctcgtggtgctcggcttcttcctccggtaccgcatcctccacccggtgcccgacgccatcccgctgtggctcacctccatcatctgcgagatctggttcgccgtgtcgtggatcctcgaccagttccccaagtggtacccgatcgacagggagacctacctcgaccgcctctccctccgctacgagcgcgagggggagccgtcgctgctgtcggcggtggacctgttcgtcagcacggtggatccgctcaaggagccgccgctggtcacggccaacaccgtgctgtccatcctcgccgtcgactaccccgtcgacaaggtgtcctgctacgtctccgacgacggcgcgtccatgctcacgttcgagtcgctgtcggagacggcggagttcgcccgcaagtgggtgccattctgcaagaagttcagcatcgagccccgcgccccggagttctacttctcccagaaggtcgactacctcaaggacaaggtccatcccaacttcgtccaggagcgccgcgccatgaaggtaatatcgatcgccatcgtcattgctgattctgtaggagcttgacgaagagctaactgttgtgggggcattggcatttgatcgagcagagagagtacgaggagttcaaggtgaggatcaacgcgctggtggcgaaggcgcagaaggtgccggcggaagggtggatcatgaaggacgggacgccatggccggggaacaacacccgcgaccacccgggcatgatccaggtgttcctcggccacagcggcggccacgacaccgagggcaacgagctcccccgcctcgtctacgtctcccgtgagaagcgccccggcttccagcaccacaagaaggccggcgccatgaacgccctcgtacgccacgccaccacatccttcatcttcaaaacaacacaactctgctcgatttgatcgaaatttctggtttgcttggcagattcgtgtgtcggccgtgctgacgaacgcgccgttcatgctcaacttggattgcgatcactacatcaacaacagcaaggccatcagggaggcgatgtgcttcctcatggatccgcaggtcggacggaaggtttgctacgtgcagttcccgcagaggttcgacggcatcgacgtccacgaccgatacgccaaccgcaacaccgtcttcttcgacgtgagctctctcccacacaaactagatgaatatatacaatcttgaaaaatccagagcaaatcacgatcgatcgagcaatgtgactgaaattttgtgtggtgcgttcttggtgagatcatcagatcaacatgaaggggcttgatgggatccagggcccggtgtacgtcgggacagggtgcgtgttcaggcggcaggcgctgtacggatacaacccacccaagggacccaagaggcccaagatggtgacctgcgactgctgcccttgcttcgggaggaagaagcggaagcacggcaaggacggcctcccggaggccgtcgccgccgacggcggtgagctcccaaattcagagctaagaagaaattatggtgtgagaagattttcagctgatggaataatgtaagtttgtgtgtggtggatcagggatggacagcgacaaggagatgctcatgtcgcagatgaacttcgagaagcggttcgggcagtcggcggcgttcgtgacgtcgacgctgatggaggaaggcggcgtcccgccgtcgtccagccccgccgcgctcctcaaggaggccatccatgtcatcagctgcggctacgaggacaagaccgactggggtctcgaggtaaccaccgttatcagacactaagccatattaccattgagtgagtttgtggtgtgaacgccatggatgtgtgtgatgcagctggggtggatctacgggtcgatcacggaggacatcctaacggggttcaagatgcactgccgcgggtggaggtcggtgtactgcatgccgaagagggcggcgttcaaggggtcagcgccgatcaacctatctgaccgtctcaaccaggtgctccggtgggcgctcggctccgtcgagatcttcttcagccggcacagcccgctcctctacggctacaagaacggcaacctcaagtggctcgagcgcttctcctacatcaacaccaccatctaccccttcacttctctccccctcctcgcctactgcaccctacccgccgtctgcctcctcaccggcaagttcatcatgcctccggtcagtcccatcccatcctgccatcaattgctcctcttcttccatggattttcccgccaaaactgaagtttcaaatgttctgaaccttttcttggtgatgcagattagcacgtttgcgagtttgttcttcatcgcgctcttcatctccatcttcgcgacgggcatcctggagatgaggtggagcggggtgagcatcgaggagtggtggaggaacgagcagttctgggtcatcggcggcgtgtcggcgcacctgttcgcggtggtgcagggcctgctcaaggtgctggccgggatcgacaccaacttcaccgtcacgtccaaggccaccggagacgaggacgacgagttcgcggagctctacgccttcaagtggaccaccctcctcatcccgcccaccacgctgctcatcctcaacatcatcggcgtcgtcgccggcgtctccgacgccatcaacaacggctccgaggcgtggggcccgctcttcgggaagctcttcttcgccttctgggtcatcgtccacctctaccccttcctcaaggggctcatggggaggcagaaccggacgcccaccattgttgtcatctggtccgtgctgctcgcctccatcttttccttgctctgggtcaggattgatcccttcaccatcaaggccaggggccctgacgtcaggcagtgcggcatcaactgctgagagagggcgtcagcgatttctgaagatttgtgcataggggggcagcaagaagatcgatttgtaaaagttttgtattgctcgtgttgagttcgtgctatgttcttctgtaatattttgggacccagaaatggtttaaacttttgaagagggaatggacagatggccatatttgaatgttaagcaacaagggctggtattctcactccatgtttgttagattttttgcagctgtgttctcattgctcctactagggatatatgggtaatttggaccaaaccatgggcattaatgtagcttaaaagtatatgaatcgatttggtcaagggctgaagtaatattcctacaaggaatatattcagattgcagc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001069742.1 RefSeq:Os09g0422500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 9]]
[[Category:Chromosome 9]]

Os09g0422500

2014-06-10T06:20:45Z

Haoyajing cathy:

The Os09g0422500 gene can be called by another gene symbols like BC6 and OsCESA9 which similar to cellulose synthase .The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle phenotype in the culm and plays an important role in cell wall biosynthesis and plant growth.

==Annotated Information==
===Introduction===
A genomic fragment containing the mutant Bc6 gene (9374 bp), including 4.8 kbp of upstream sequence and 492 bp downstream, was digested with HindIII and subcloned into the binary vector pBIGRZ to yield the mutant construct, gBc6/pBIGRZ. The wild-type BC6 gene construct, gBC6/pBIGRZ, was generated by changing the mutated nucleotide at position 7112 from G to wild-type A by PCR mutagenesis with primers, PM-F1 (5′-GCAGTTCCCGCAGAGGTTCGACGGC-3′) and PM-R1 (5′-ATCGACGTCCACGACCGATACGCC-3′). These constructs were introduced into the wild-type plant, T65, by an Agrobacterium (Rhizobium radiobacter)-mediated method using strain EHA101. The T2 generation of transgenic plant was used for cell wall analysis.
[[File: haoyajing.jpg]]

1.Promoter

PromoterBC6:β-glucuronidase (pBC6:GUS) activity was assayed according to the method of Kosugi et al.The 4.8 kbp promoter region of BC6 was amplified by PCR using specific primers, gBc6-F1 and pBC6-R1 (5′-GAGGATCCATGGCCGCGCAACAACGGCCGG-3′), and fused with the GUS gene in the pBIGRZ vector, yielding pBC6:GUS. The nucleotide sequence of the construct was confirmed before transformation into the wild-type plant, T65, as described above. Culms, leaves, nodes, and seedlings of the transgenic plants harbouring the pBC6:GUS gene were cut into pieces, fixed in 5% (w/v) agar, and hand-sectioned. Sections from the transgenic plants were stained with a solution containing 0.5 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 50 mM phosphate buffer (pH 7.4) at 37 °C for 24 h, and observed under a microscope (Eclipse E400).

2.Quantitative analysis of BC1 and BC6 mRNAs

Relative amounts of BC1, BC3, BC6, OsCesA4, and OsCesA7 mRNA were estimated by quantitative RT-PCR. Single-stranded cDNA was synthesized from total RNA of the tissues or organs using oligo(dT)12–18 primer. The following specific primers were designed using the Primer3 program (http://frodo.wi.mit.edu/): for BC1 (Os03g0416200), BC1-RTP-F1 (5′-CGCATGAACTACACCCAGTG-3′) and BC1-RTP-R1 (5′-TCCATGAGCAGGTCGTTGTA-3′); for BC3 (Os02g0738900), BC3-RTP-F1 (5′-GGCCGAAACGATGAGATTTA-3′) and BC3-RTP-R1 (5′- AACATCAGCAGCTTGCATTG-3′); for BC6 (Os09g0422500), BC6-RTP-F1 (5′-TTAGCACGTTTGCGAGTTTG-3′) and BC6-RTP-R1 (5′-GAACTCGTCGTCCTCGTCTC-3′); for OsCesA4 (Os01g0750300), OsCesA4-RTP-F1 (5′-CTAATGCGACGAAGACGATG-3′) and OsCesA4-RTP-R1 (5′-GATTTAACGGTGCCCTCTCA-3′); for OsCesA7 (Os10g0467800), OsCesA7-RTP-F1 (5′-TCCATCTTCTCCCTCGTCTG-3′) and OsCesA7-RTP-R1 (5′-GAATCATCCATCCGGTCATC-3′); and for ACTIN1 (Os03g0718100), ACT1-RTP-F1 (5′-TTCCTACATCGCCCTGGACT-3′) and ACT1-RTP-R1 (5′-AGCCTTGGCAATCCACATCT-3′). The PCR was performed with a SYBR Premix Ex Taq kit under the following conditions: 10 s denaturing at 95 °C, 30 s annealing at 60 °C, and 20 s amplification at 72 C, 40 cycles. The PCR products were detected with Opticon 2 , and the mRNA amounts relative to ACTIN1 mRNA were calculated.

===Function===
1;This study characterizes a brittle culm (bc88) mutant of rice (Oryza sativa L.) obtained by ethylene methylsulfonate (EMS)-induced mutagenesis of Wuyunjing 7. The bc88 mutant exhibits a diversity of pleiotropic phenotypes, including brittle culm at the whole-plant growth stages, withered leaf tips at the seedling stage, and 18-d delay in heading date at the mature stage. Genetic analysis indicates that the bc88 mutant is controlled by a single recessive nuclear gene. The mutated bc88 gene isolated by map-based cloning contains only one point mutation in the 5th exon relative to its wild-type BC88 (LOC_Os09g25490 and Os09g0422500), leading to an amino acid change from P to L in bc88 plants. Alignment of the putative protein sequence with its homologs indicates that the mutation is located in the conserved region of the sequence. Detection of the transcription level of BC88 in rice plants shows that the expression level of BC88 is higher in spikes and culms than in leaves, roots, and leaf sheaths. These contribute to understanding of the molecular mechanism of cellulose synthesis. The target gene BC88 can be a useful tool in molecular marker-assisted selection for rice culm trait breeding.

2;Rice is a model organism in poaceae plants to study cell wall biosynthesis. In this study, a mutant S1-60 isolated from an EMS mutagenized japonica cultivar Nipponbare, is characterized by brittle culms that can be easily broken by bending. The reduction in mechanical strength was due to defect in thickening of the sclerenchyma cell wall. The amount of cellulose in S1-60 culms was reduced to 44.7% of that of wild-type plants. Besides, the mutant also exhibited pleiotropic phenotypes, such as dwarfism and partial sterility. Genetic analysis and map-based cloning showed that all the phenotype of S1-60 mutant was caused by a recessive point mutation in the OsCESA9 gene, which encodes the cellulose synthase A subunit 9. This yet uncharacterized missense mutation changed the highly conserved G905 to D at the beginning of the fifth transmembrane domain. The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle phenotype in the culm. These results indicate that OsCESA9 plays an important role in cell wall biosynthesis and plant growth.

3;According to the MSU Rice Genome Annotation Release 7 (http://rice.plantbiology.msu.edu), there are 13 predicted open reading frames (ORFs) within the 118 kb fine mapping interval , including 7 ORFs with known biochemical functions, 5 ORFs encoding expressed hypothetical protein and 1 transposon . Among them, ORF3 (TIGR ID: LOC_Os09g25490) encoding cellulose synthase A catalytic subunit 9 (OsCESA9) was considered as the priority candidate gene, given that the amount of cellulose was reduced dramatically in S1-60 culm. Therefore, we sequenced and compared the mutant and wild type alleles of the OsCESA9 gene, which has 4643 bp in length and 11 exons and 10 introns. One base pair substitution was found in the last exon, changing GGC to GAC and the encoded amino acid from glycine to aspartic acid at the 905th position .The CESA9 protein has 1056 amino acids in length and eight putative transmembrane domains (TMDs), with two near the amino terminus and six clustered near the carboxyl terminus . There is a RING-type zinc finger in the N-terminal region, which might mediate the interaction between CESA9 and other CESA subunits. A large central domain containing two highly conserved motifs DXD and Q/RXXRW is required for the catalytic activity of the enzyme. The missense mutation in S1-60 occurs at the beginning of the fifth TMD, which might affect the plasma membrane localization of CESA9.

4;The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.

===Expression===
We examined the expression level of OsCESA9 in various rice organs by both semi-quantitative and quantitative RT-PCR. The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle culm phenotype in the S1-60 mutant. The OsCESA9 gene is also expressed in panicle at mature stage. However, the expression level of OsCESA9 was relatively low at seedling stage, no matter in root, leaf blade or leaf sheath. This result showed the OsCESA9 gene mainly participates in the synthesis of secondary cell wall in mechanical tissue at late development stage.

The pattern of expression of BC6 was assessed by analysing T65 plants transformed with pBC6:GUS, a binary vector in which the 4.8 kbp region upstream of the BC6 gene was fused with the GUS reporter gene. BC6 promoter activity was detected in leaves, culms, and nodes, with relatively strong expression in culm vascular bundles 2 weeks after heading.Promoter activity was also observed in young tissues such as developing leaves. Although Bc6 mutation appeared to reduce cell wall thickness in sclerenchymal cells , promoter activity was not detected in developed sclerenchymal cells .It is possible that the reporter gene activity did not completely mirror the expression of the BC6 gene product, OsCesA9 protein. These patterns of BC6 promoter-driven gene expression were similar to the expression pattern of BC1 demonstrated by in situ hybridization .

In Arabidopsis, the COBL4 gene is co-expressed with the secondary cell wall-specific CesA genes and is presumed to play a role in the cellulose synthesis of secondary cell walls, although the precise molecular functions of COBRA-like proteins remain unclear .To examine the relationship between the secondary cell wall-specific CesA and COBRA-like proteins in rice, BC6 and BC1 mRNAs were quantitated in several tissues. Consistent with the results of the pBC6:GUS analysis, relatively high levels of BC6 mRNA were detected in culms and nodes.The level of Bc6 mRNA in roots was relatively low. Indeed, the brittle phenotype in roots was not clear compared with those in culms and leaves.Despite the apparent brittle phenotype in the leaves of Bc6 mutants, the level was low in both leaf blades and sheaths. Importantly, both BC6 and BC1 were highly expressed in culms, nodes, and flowers .These results indicated that BC6 and BC1 are co-expressed during development of secondary cell walls. On the other hand, the expression of BC6 was not related to that of BC3, suggesting that BC6 and BC3 are differently regulated.

The effect of the mutation on the expression of Bc6 was also examined in developing leaf blades. Bc6 mutants showed accumulation of BC6 mRNA comparable with T65.Furthermore, Bc6 mutation barely influenced the mRNA levels of other CesA genes, OsCesA4 and OsCesA7, which are expected to participate in cellulose synthesis in secondary cell walls, together with BC6 (OsCesA9).

===Evolution===
Please input evolution information here.

You can also add sub-section(s) at will.

==Labs working on this gene==
1. College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao 266109, China

2. National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

3. Shandong Rice Research Institute, Jinan 250100, China

4. College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China

5. Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan

6. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China;

7. State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China

==References==
1.A missense mutation in the transmembrane domain of CESA9 affects cell wall biosynthesis and plant growth in rice. Wang D, et al. Plant Sci, 2012 Nov. PMID 23017906

2.Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls. Kotake T, et al. J Exp Bot, 2011 Mar. PMID 21209026, Free PMC Article

3.The Rice Annotation Project Database (RAP-DB): 2008 update. Rice Annotation Project, et al. Nucleic Acids Res, 2008 Jan. PMID 18089549, Free PMC Article

4.Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana. Rice Annotation Project, et al. Genome Res, 2007 Feb. PMID 17210932, Free PMC Article

5.The Rice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp. japonica genome information. Ohyanagi H, et al. Nucleic Acids Res, 2006 Jan 1. PMID 16381971, Free PMC Article
==Structured Information==
{{JaponicaGene|
GeneName = Os09g0422500|
Description = Similar to Cellulose synthase (Fragment)|
Version = NM_001069742.1 GI:115479226 GeneID:4347093|
Length = 4574 bp|
Definition = Oryza sativa Japonica Group Os09g0422500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 9|Chromosome 9]]|
AP = Chromosome 9:15935031..15939604|
CDS = 15935124..15935394,15935500..15935735,15935831..15935927,15936019..15936145,15936232..15936844 ,15936934..15937197,15937280..15937492,15937615..15937822,15937914..15938110 ,15938192..15938545,15938649..15939236|
GCID = <gbrowseImage1>
name=NC_008402:15935031..15939604
source=RiceChromosome09
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008402:15935031..15939604
source=RiceChromosome09
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggaggcgagcgccgggctggtggccgggtcgcacaaccggaacgagctggtgctgatccgggggcacgaggagcccaagccgctgcgggcgctgagcgggcaggtgtgcgagatatgcggcgacgaggtcggccgcaccgtcgacggcgacctcttcgtcgcctgcaacgagtgcggcttcccggtgtgccgcccctgctacgagtacgagcgccgcgagggcacccagaactgcccccagtgcaagacccgctacaagcgcctcaaggggagcccgagggtgcccggggacgaggacgaggaggacattgacgacctggagcacgagttcaacatcgacgacgagaagcagaagcagctgcagcaggatcaggatggcatgcagaacagccacatcaccgaggcgatgctgcacggcaagatgagctacgggaggggccccgacgacggcgacggcaacagcaccccgctcccgccgatcatcaccggcgctcgctccgtcccggtgagcggggagttcccgatatcgaacagccatggccatggcgagttctcctcttccctgcacaagcgcatccacccctacccggtgtctgagccagggagtgcaaagtgggacgagaagaaagaggtgagctggaaggagaggatggacgactggaaatccaagcagggcatcgtcgccggcggcgcccccgatcccgacgactacgacgccgacgtcccactgaacgacgaggcgaggcagccgctgtcgaggaaggtgtcgatcgcgtcgagcaaggtgaacccgtaccggatggtgatcatcctccgtctcgtggtgctcggcttcttcctccggtaccgcatcctccacccggtgcccgacgccatcccgctgtggctcacctccatcatctgcgagatctggttcgccgtgtcgtggatcctcgaccagttccccaagtggtacccgatcgacagggagacctacctcgaccgcctctccctccgctacgagcgcgagggggagccgtcgctgctgtcggcggtggacctgttcgtcagcacggtggatccgctcaaggagccgccgctggtcacggccaacaccgtgctgtccatcctcgccgtcgactaccccgtcgacaaggtgtcctgctacgtctccgacgacggcgcgtccatgctcacgttcgagtcgctgtcggagacggcggagttcgcccgcaagtgggtgccattctgcaagaagttcagcatcgagccccgcgccccggagttctacttctcccagaaggtcgactacctcaaggacaaggtccatcccaacttcgtccaggagcgccgcgccatgaagagagagtacgaggagttcaaggtgaggatcaacgcgctggtggcgaaggcgcagaaggtgccggcggaagggtggatcatgaaggacgggacgccatggccggggaacaacacccgcgaccacccgggcatgatccaggtgttcctcggccacagcggcggccacgacaccgagggcaacgagctcccccgcctcgtctacgtctcccgtgagaagcgccccggcttccagcaccacaagaaggccggcgccatgaacgccctcattcgtgtgtcggccgtgctgacgaacgcgccgttcatgctcaacttggattgcgatcactacatcaacaacagcaaggccatcagggaggcgatgtgcttcctcatggatccgcaggtcggacggaaggtttgctacgtgcagttcccgcagaggttcgacggcatcgacgtccacgaccgatacgccaaccgcaacaccgtcttcttcgacatcaacatgaaggggcttgatgggatccagggcccggtgtacgtcgggacagggtgcgtgttcaggcggcaggcgctgtacggatacaacccacccaagggacccaagaggcccaagatggtgacctgcgactgctgcccttgcttcgggaggaagaagcggaagcacggcaaggacggcctcccggaggccgtcgccgccgacggcgggatggacagcgacaaggagatgctcatgtcgcagatgaacttcgagaagcggttcgggcagtcggcggcgttcgtgacgtcgacgctgatggaggaaggcggcgtcccgccgtcgtccagccccgccgcgctcctcaaggaggccatccatgtcatcagctgcggctacgaggacaagaccgactggggtctcgagctggggtggatctacgggtcgatcacggaggacatcctaacggggttcaagatgcactgccgcgggtggaggtcggtgtactgcatgccgaagagggcggcgttcaaggggtcagcgccgatcaacctatctgaccgtctcaaccaggtgctccggtgggcgctcggctccgtcgagatcttcttcagccggcacagcccgctcctctacggctacaagaacggcaacctcaagtggctcgagcgcttctcctacatcaacaccaccatctaccccttcacttctctccccctcctcgcctactgcaccctacccgccgtctgcctcctcaccggcaagttcatcatgcctccgattagcacgtttgcgagtttgttcttcatcgcgctcttcatctccatcttcgcgacgggcatcctggagatgaggtggagcggggtgagcatcgaggagtggtggaggaacgagcagttctgggtcatcggcggcgtgtcggcgcacctgttcgcggtggtgcagggcctgctcaaggtgctggccgggatcgacaccaacttcaccgtcacgtccaaggccaccggagacgaggacgacgagttcgcggagctctacgccttcaagtggaccaccctcctcatcccgcccaccacgctgctcatcctcaacatcatcggcgtcgtcgccggcgtctccgacgccatcaacaacggctccgaggcgtggggcccgctcttcgggaagctcttcttcgccttctgggtcatcgtccacctctaccccttcctcaaggggctcatggggaggcagaaccggacgcccaccattgttgtcatctggtccgtgctgctcgcctccatcttttccttgctctgggtcaggattgatcccttcaccatcaaggccaggggccctgacgtcaggcagtgcggcatcaactgctga</cdnaseq>|
AA = <aaseq>MEASAGLVAGSHNRNELVLIRGHEEPKPLRALSGQVCEICGDEV GRTVDGDLFVACNECGFPVCRPCYEYERREGTQNCPQCKTRYKRLKGSPRVPGDEDEE DIDDLEHEFNIDDEKQKQLQQDQDGMQNSHITEAMLHGKMSYGRGPDDGDGNSTPLPP IITGARSVPVSGEFPISNSHGHGEFSSSLHKRIHPYPVSEPGSAKWDEKKEVSWKERM DDWKSKQGIVAGGAPDPDDYDADVPLNDEARQPLSRKVSIASSKVNPYRMVIILRLVV LGFFLRYRILHPVPDAIPLWLTSIICEIWFAVSWILDQFPKWYPIDRETYLDRLSLRY EREGEPSLLSAVDLFVSTVDPLKEPPLVTANTVLSILAVDYPVDKVSCYVSDDGASML TFESLSETAEFARKWVPFCKKFSIEPRAPEFYFSQKVDYLKDKVHPNFVQERRAMKRE YEEFKVRINALVAKAQKVPAEGWIMKDGTPWPGNNTRDHPGMIQVFLGHSGGHDTEGN ELPRLVYVSREKRPGFQHHKKAGAMNALIRVSAVLTNAPFMLNLDCDHYINNSKAIRE AMCFLMDPQVGRKVCYVQFPQRFDGIDVHDRYANRNTVFFDINMKGLDGIQGPVYVGT GCVFRRQALYGYNPPKGPKRPKMVTCDCCPCFGRKKRKHGKDGLPEAVAADGGMDSDK EMLMSQMNFEKRFGQSAAFVTSTLMEEGGVPPSSSPAALLKEAIHVISCGYEDKTDWG LELGWIYGSITEDILTGFKMHCRGWRSVYCMPKRAAFKGSAPINLSDRLNQVLRWALG SVEIFFSRHSPLLYGYKNGNLKWLERFSYINTTIYPFTSLPLLAYCTLPAVCLLTGKF IMPPISTFASLFFIALFISIFATGILEMRWSGVSIEEWWRNEQFWVIGGVSAHLFAVV QGLLKVLAGIDTNFTVTSKATGDEDDEFAELYAFKWTTLLIPPTTLLILNIIGVVAGV SDAINNGSEAWGPLFGKLFFAFWVIVHLYPFLKGLMGRQNRTPTIVVIWSVLLASIFS LLWVRIDPFTIKARGPDVRQCGINC</aaseq>|
DNA = <dnaseqindica>94..364#470..705#801..897#989..1115#1202..1814#1904..2167#2250..2462#2585..2792#2884..3080#3162..3515#3619..4206#agcgatcgatcgcccttcctcctcctcctctcctccttcctgcgtcgcctccctgatcagccgcagctcgttgccggccgttgttgcgcggccatggaggcgagcgccgggctggtggccgggtcgcacaaccggaacgagctggtgctgatccgggggcacgaggagcccaagccgctgcgggcgctgagcgggcaggtgtgcgagatatgcggcgacgaggtcggccgcaccgtcgacggcgacctcttcgtcgcctgcaacgagtgcggcttcccggtgtgccgcccctgctacgagtacgagcgccgcgagggcacccagaactgcccccagtgcaagacccgctacaagcgcctcaagggtaaaaaaacacactcacatcacgctacgcccttgataccgcgatcgcgaggtttccgttgattgatctctaatggcgatggtggtggtggtggttttgttacagggagcccgagggtgcccggggacgaggacgaggaggacattgacgacctggagcacgagttcaacatcgacgacgagaagcagaagcagctgcagcaggatcaggatggcatgcagaacagccacatcaccgaggcgatgctgcacggcaagatgagctacgggaggggccccgacgacggcgacggcaacagcaccccgctcccgccgatcatcaccggcgctcgctccgtcccggtacacacaacacacctcaccccactcacaccaaattctctccctcttctcacctcaacatgttcacgaaatgttctttgtaaaaaaaaattcaggtgagcggggagttcccgatatcgaacagccatggccatggcgagttctcctcttccctgcacaagcgcatccacccctacccggtgtctgagccaggtagtataacgaattcactacactaattaatcaatgttcttgatgaacacacattgatcatctcaatcatctaccgatgaattctgaccagggagtgcaaagtgggacgagaagaaagaggtgagctggaaggagaggatggacgactggaaatccaagcagggcatcgtcgccggcggcgcccccgatcccgacgactacgacgccgacgtcccactgtacgcaatcctcagctgagcagcttacactgatcatgcatgacaactagtatattgatcatgcctgaactctctgtggcttgcaggaacgacgaggcgaggcagccgctgtcgaggaaggtgtcgatcgcgtcgagcaaggtgaacccgtaccggatggtgatcatcctccgtctcgtggtgctcggcttcttcctccggtaccgcatcctccacccggtgcccgacgccatcccgctgtggctcacctccatcatctgcgagatctggttcgccgtgtcgtggatcctcgaccagttccccaagtggtacccgatcgacagggagacctacctcgaccgcctctccctccgctacgagcgcgagggggagccgtcgctgctgtcggcggtggacctgttcgtcagcacggtggatccgctcaaggagccgccgctggtcacggccaacaccgtgctgtccatcctcgccgtcgactaccccgtcgacaaggtgtcctgctacgtctccgacgacggcgcgtccatgctcacgttcgagtcgctgtcggagacggcggagttcgcccgcaagtgggtgccattctgcaagaagttcagcatcgagccccgcgccccggagttctacttctcccagaaggtcgactacctcaaggacaaggtccatcccaacttcgtccaggagcgccgcgccatgaaggtaatatcgatcgccatcgtcattgctgattctgtaggagcttgacgaagagctaactgttgtgggggcattggcatttgatcgagcagagagagtacgaggagttcaaggtgaggatcaacgcgctggtggcgaaggcgcagaaggtgccggcggaagggtggatcatgaaggacgggacgccatggccggggaacaacacccgcgaccacccgggcatgatccaggtgttcctcggccacagcggcggccacgacaccgagggcaacgagctcccccgcctcgtctacgtctcccgtgagaagcgccccggcttccagcaccacaagaaggccggcgccatgaacgccctcgtacgccacgccaccacatccttcatcttcaaaacaacacaactctgctcgatttgatcgaaatttctggtttgcttggcagattcgtgtgtcggccgtgctgacgaacgcgccgttcatgctcaacttggattgcgatcactacatcaacaacagcaaggccatcagggaggcgatgtgcttcctcatggatccgcaggtcggacggaaggtttgctacgtgcagttcccgcagaggttcgacggcatcgacgtccacgaccgatacgccaaccgcaacaccgtcttcttcgacgtgagctctctcccacacaaactagatgaatatatacaatcttgaaaaatccagagcaaatcacgatcgatcgagcaatgtgactgaaattttgtgtggtgcgttcttggtgagatcatcagatcaacatgaaggggcttgatgggatccagggcccggtgtacgtcgggacagggtgcgtgttcaggcggcaggcgctgtacggatacaacccacccaagggacccaagaggcccaagatggtgacctgcgactgctgcccttgcttcgggaggaagaagcggaagcacggcaaggacggcctcccggaggccgtcgccgccgacggcggtgagctcccaaattcagagctaagaagaaattatggtgtgagaagattttcagctgatggaataatgtaagtttgtgtgtggtggatcagggatggacagcgacaaggagatgctcatgtcgcagatgaacttcgagaagcggttcgggcagtcggcggcgttcgtgacgtcgacgctgatggaggaaggcggcgtcccgccgtcgtccagccccgccgcgctcctcaaggaggccatccatgtcatcagctgcggctacgaggacaagaccgactggggtctcgaggtaaccaccgttatcagacactaagccatattaccattgagtgagtttgtggtgtgaacgccatggatgtgtgtgatgcagctggggtggatctacgggtcgatcacggaggacatcctaacggggttcaagatgcactgccgcgggtggaggtcggtgtactgcatgccgaagagggcggcgttcaaggggtcagcgccgatcaacctatctgaccgtctcaaccaggtgctccggtgggcgctcggctccgtcgagatcttcttcagccggcacagcccgctcctctacggctacaagaacggcaacctcaagtggctcgagcgcttctcctacatcaacaccaccatctaccccttcacttctctccccctcctcgcctactgcaccctacccgccgtctgcctcctcaccggcaagttcatcatgcctccggtcagtcccatcccatcctgccatcaattgctcctcttcttccatggattttcccgccaaaactgaagtttcaaatgttctgaaccttttcttggtgatgcagattagcacgtttgcgagtttgttcttcatcgcgctcttcatctccatcttcgcgacgggcatcctggagatgaggtggagcggggtgagcatcgaggagtggtggaggaacgagcagttctgggtcatcggcggcgtgtcggcgcacctgttcgcggtggtgcagggcctgctcaaggtgctggccgggatcgacaccaacttcaccgtcacgtccaaggccaccggagacgaggacgacgagttcgcggagctctacgccttcaagtggaccaccctcctcatcccgcccaccacgctgctcatcctcaacatcatcggcgtcgtcgccggcgtctccgacgccatcaacaacggctccgaggcgtggggcccgctcttcgggaagctcttcttcgccttctgggtcatcgtccacctctaccccttcctcaaggggctcatggggaggcagaaccggacgcccaccattgttgtcatctggtccgtgctgctcgcctccatcttttccttgctctgggtcaggattgatcccttcaccatcaaggccaggggccctgacgtcaggcagtgcggcatcaactgctgagagagggcgtcagcgatttctgaagatttgtgcataggggggcagcaagaagatcgatttgtaaaagttttgtattgctcgtgttgagttcgtgctatgttcttctgtaatattttgggacccagaaatggtttaaacttttgaagagggaatggacagatggccatatttgaatgttaagcaacaagggctggtattctcactccatgtttgttagattttttgcagctgtgttctcattgctcctactagggatatatgggtaatttggaccaaaccatgggcattaatgtagcttaaaagtatatgaatcgatttggtcaagggctgaagtaatattcctacaaggaatatattcagattgcagc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001069742.1 RefSeq:Os09g0422500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 9]]
[[Category:Chromosome 9]]

File:Haoyajing.jpg

2014-06-10T06:20:06Z

Haoyajing cathy: uploaded a new version of "File:Haoyajing.jpg"

Os09g0422500

2014-06-10T06:18:26Z

Haoyajing cathy:

The Os09g0422500 gene can be called by another gene symbols like BC6 and OsCESA9 which similar to cellulose synthase .The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle phenotype in the culm and plays an important role in cell wall biosynthesis and plant growth.

==Annotated Information==
===Introduction===
A genomic fragment containing the mutant Bc6 gene (9374 bp), including 4.8 kbp of upstream sequence and 492 bp downstream, was digested with HindIII and subcloned into the binary vector pBIGRZ to yield the mutant construct, gBc6/pBIGRZ. The wild-type BC6 gene construct, gBC6/pBIGRZ, was generated by changing the mutated nucleotide at position 7112 from G to wild-type A by PCR mutagenesis with primers, PM-F1 (5′-GCAGTTCCCGCAGAGGTTCGACGGC-3′) and PM-R1 (5′-ATCGACGTCCACGACCGATACGCC-3′). These constructs were introduced into the wild-type plant, T65, by an Agrobacterium (Rhizobium radiobacter)-mediated method using strain EHA101. The T2 generation of transgenic plant was used for cell wall analysis.[[File: haoyajing.jpg]]

1.Promoter

PromoterBC6:β-glucuronidase (pBC6:GUS) activity was assayed according to the method of Kosugi et al.The 4.8 kbp promoter region of BC6 was amplified by PCR using specific primers, gBc6-F1 and pBC6-R1 (5′-GAGGATCCATGGCCGCGCAACAACGGCCGG-3′), and fused with the GUS gene in the pBIGRZ vector, yielding pBC6:GUS. The nucleotide sequence of the construct was confirmed before transformation into the wild-type plant, T65, as described above. Culms, leaves, nodes, and seedlings of the transgenic plants harbouring the pBC6:GUS gene were cut into pieces, fixed in 5% (w/v) agar, and hand-sectioned. Sections from the transgenic plants were stained with a solution containing 0.5 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 50 mM phosphate buffer (pH 7.4) at 37 °C for 24 h, and observed under a microscope (Eclipse E400).

2.Quantitative analysis of BC1 and BC6 mRNAs

Relative amounts of BC1, BC3, BC6, OsCesA4, and OsCesA7 mRNA were estimated by quantitative RT-PCR. Single-stranded cDNA was synthesized from total RNA of the tissues or organs using oligo(dT)12–18 primer. The following specific primers were designed using the Primer3 program (http://frodo.wi.mit.edu/): for BC1 (Os03g0416200), BC1-RTP-F1 (5′-CGCATGAACTACACCCAGTG-3′) and BC1-RTP-R1 (5′-TCCATGAGCAGGTCGTTGTA-3′); for BC3 (Os02g0738900), BC3-RTP-F1 (5′-GGCCGAAACGATGAGATTTA-3′) and BC3-RTP-R1 (5′- AACATCAGCAGCTTGCATTG-3′); for BC6 (Os09g0422500), BC6-RTP-F1 (5′-TTAGCACGTTTGCGAGTTTG-3′) and BC6-RTP-R1 (5′-GAACTCGTCGTCCTCGTCTC-3′); for OsCesA4 (Os01g0750300), OsCesA4-RTP-F1 (5′-CTAATGCGACGAAGACGATG-3′) and OsCesA4-RTP-R1 (5′-GATTTAACGGTGCCCTCTCA-3′); for OsCesA7 (Os10g0467800), OsCesA7-RTP-F1 (5′-TCCATCTTCTCCCTCGTCTG-3′) and OsCesA7-RTP-R1 (5′-GAATCATCCATCCGGTCATC-3′); and for ACTIN1 (Os03g0718100), ACT1-RTP-F1 (5′-TTCCTACATCGCCCTGGACT-3′) and ACT1-RTP-R1 (5′-AGCCTTGGCAATCCACATCT-3′). The PCR was performed with a SYBR Premix Ex Taq kit under the following conditions: 10 s denaturing at 95 °C, 30 s annealing at 60 °C, and 20 s amplification at 72 C, 40 cycles. The PCR products were detected with Opticon 2 , and the mRNA amounts relative to ACTIN1 mRNA were calculated.

===Function===
1;This study characterizes a brittle culm (bc88) mutant of rice (Oryza sativa L.) obtained by ethylene methylsulfonate (EMS)-induced mutagenesis of Wuyunjing 7. The bc88 mutant exhibits a diversity of pleiotropic phenotypes, including brittle culm at the whole-plant growth stages, withered leaf tips at the seedling stage, and 18-d delay in heading date at the mature stage. Genetic analysis indicates that the bc88 mutant is controlled by a single recessive nuclear gene. The mutated bc88 gene isolated by map-based cloning contains only one point mutation in the 5th exon relative to its wild-type BC88 (LOC_Os09g25490 and Os09g0422500), leading to an amino acid change from P to L in bc88 plants. Alignment of the putative protein sequence with its homologs indicates that the mutation is located in the conserved region of the sequence. Detection of the transcription level of BC88 in rice plants shows that the expression level of BC88 is higher in spikes and culms than in leaves, roots, and leaf sheaths. These contribute to understanding of the molecular mechanism of cellulose synthesis. The target gene BC88 can be a useful tool in molecular marker-assisted selection for rice culm trait breeding.

2;Rice is a model organism in poaceae plants to study cell wall biosynthesis. In this study, a mutant S1-60 isolated from an EMS mutagenized japonica cultivar Nipponbare, is characterized by brittle culms that can be easily broken by bending. The reduction in mechanical strength was due to defect in thickening of the sclerenchyma cell wall. The amount of cellulose in S1-60 culms was reduced to 44.7% of that of wild-type plants. Besides, the mutant also exhibited pleiotropic phenotypes, such as dwarfism and partial sterility. Genetic analysis and map-based cloning showed that all the phenotype of S1-60 mutant was caused by a recessive point mutation in the OsCESA9 gene, which encodes the cellulose synthase A subunit 9. This yet uncharacterized missense mutation changed the highly conserved G905 to D at the beginning of the fifth transmembrane domain. The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle phenotype in the culm. These results indicate that OsCESA9 plays an important role in cell wall biosynthesis and plant growth.

3;According to the MSU Rice Genome Annotation Release 7 (http://rice.plantbiology.msu.edu), there are 13 predicted open reading frames (ORFs) within the 118 kb fine mapping interval , including 7 ORFs with known biochemical functions, 5 ORFs encoding expressed hypothetical protein and 1 transposon . Among them, ORF3 (TIGR ID: LOC_Os09g25490) encoding cellulose synthase A catalytic subunit 9 (OsCESA9) was considered as the priority candidate gene, given that the amount of cellulose was reduced dramatically in S1-60 culm. Therefore, we sequenced and compared the mutant and wild type alleles of the OsCESA9 gene, which has 4643 bp in length and 11 exons and 10 introns. One base pair substitution was found in the last exon, changing GGC to GAC and the encoded amino acid from glycine to aspartic acid at the 905th position .The CESA9 protein has 1056 amino acids in length and eight putative transmembrane domains (TMDs), with two near the amino terminus and six clustered near the carboxyl terminus . There is a RING-type zinc finger in the N-terminal region, which might mediate the interaction between CESA9 and other CESA subunits. A large central domain containing two highly conserved motifs DXD and Q/RXXRW is required for the catalytic activity of the enzyme. The missense mutation in S1-60 occurs at the beginning of the fifth TMD, which might affect the plasma membrane localization of CESA9.

4;The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.

===Expression===
We examined the expression level of OsCESA9 in various rice organs by both semi-quantitative and quantitative RT-PCR. The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle culm phenotype in the S1-60 mutant. The OsCESA9 gene is also expressed in panicle at mature stage. However, the expression level of OsCESA9 was relatively low at seedling stage, no matter in root, leaf blade or leaf sheath. This result showed the OsCESA9 gene mainly participates in the synthesis of secondary cell wall in mechanical tissue at late development stage.

The pattern of expression of BC6 was assessed by analysing T65 plants transformed with pBC6:GUS, a binary vector in which the 4.8 kbp region upstream of the BC6 gene was fused with the GUS reporter gene. BC6 promoter activity was detected in leaves, culms, and nodes, with relatively strong expression in culm vascular bundles 2 weeks after heading.Promoter activity was also observed in young tissues such as developing leaves. Although Bc6 mutation appeared to reduce cell wall thickness in sclerenchymal cells , promoter activity was not detected in developed sclerenchymal cells .It is possible that the reporter gene activity did not completely mirror the expression of the BC6 gene product, OsCesA9 protein. These patterns of BC6 promoter-driven gene expression were similar to the expression pattern of BC1 demonstrated by in situ hybridization .

In Arabidopsis, the COBL4 gene is co-expressed with the secondary cell wall-specific CesA genes and is presumed to play a role in the cellulose synthesis of secondary cell walls, although the precise molecular functions of COBRA-like proteins remain unclear .To examine the relationship between the secondary cell wall-specific CesA and COBRA-like proteins in rice, BC6 and BC1 mRNAs were quantitated in several tissues. Consistent with the results of the pBC6:GUS analysis, relatively high levels of BC6 mRNA were detected in culms and nodes.The level of Bc6 mRNA in roots was relatively low. Indeed, the brittle phenotype in roots was not clear compared with those in culms and leaves.Despite the apparent brittle phenotype in the leaves of Bc6 mutants, the level was low in both leaf blades and sheaths. Importantly, both BC6 and BC1 were highly expressed in culms, nodes, and flowers .These results indicated that BC6 and BC1 are co-expressed during development of secondary cell walls. On the other hand, the expression of BC6 was not related to that of BC3, suggesting that BC6 and BC3 are differently regulated.

The effect of the mutation on the expression of Bc6 was also examined in developing leaf blades. Bc6 mutants showed accumulation of BC6 mRNA comparable with T65.Furthermore, Bc6 mutation barely influenced the mRNA levels of other CesA genes, OsCesA4 and OsCesA7, which are expected to participate in cellulose synthesis in secondary cell walls, together with BC6 (OsCesA9).

===Evolution===
Please input evolution information here.

You can also add sub-section(s) at will.

==Labs working on this gene==
1. College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao 266109, China

2. National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

3. Shandong Rice Research Institute, Jinan 250100, China

4. College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China

5. Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan

6. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China;

7. State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China

==References==
1.A missense mutation in the transmembrane domain of CESA9 affects cell wall biosynthesis and plant growth in rice. Wang D, et al. Plant Sci, 2012 Nov. PMID 23017906

2.Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls. Kotake T, et al. J Exp Bot, 2011 Mar. PMID 21209026, Free PMC Article

3.The Rice Annotation Project Database (RAP-DB): 2008 update. Rice Annotation Project, et al. Nucleic Acids Res, 2008 Jan. PMID 18089549, Free PMC Article

4.Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana. Rice Annotation Project, et al. Genome Res, 2007 Feb. PMID 17210932, Free PMC Article

5.The Rice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp. japonica genome information. Ohyanagi H, et al. Nucleic Acids Res, 2006 Jan 1. PMID 16381971, Free PMC Article
==Structured Information==
{{JaponicaGene|
GeneName = Os09g0422500|
Description = Similar to Cellulose synthase (Fragment)|
Version = NM_001069742.1 GI:115479226 GeneID:4347093|
Length = 4574 bp|
Definition = Oryza sativa Japonica Group Os09g0422500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 9|Chromosome 9]]|
AP = Chromosome 9:15935031..15939604|
CDS = 15935124..15935394,15935500..15935735,15935831..15935927,15936019..15936145,15936232..15936844 ,15936934..15937197,15937280..15937492,15937615..15937822,15937914..15938110 ,15938192..15938545,15938649..15939236|
GCID = <gbrowseImage1>
name=NC_008402:15935031..15939604
source=RiceChromosome09
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008402:15935031..15939604
source=RiceChromosome09
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggaggcgagcgccgggctggtggccgggtcgcacaaccggaacgagctggtgctgatccgggggcacgaggagcccaagccgctgcgggcgctgagcgggcaggtgtgcgagatatgcggcgacgaggtcggccgcaccgtcgacggcgacctcttcgtcgcctgcaacgagtgcggcttcccggtgtgccgcccctgctacgagtacgagcgccgcgagggcacccagaactgcccccagtgcaagacccgctacaagcgcctcaaggggagcccgagggtgcccggggacgaggacgaggaggacattgacgacctggagcacgagttcaacatcgacgacgagaagcagaagcagctgcagcaggatcaggatggcatgcagaacagccacatcaccgaggcgatgctgcacggcaagatgagctacgggaggggccccgacgacggcgacggcaacagcaccccgctcccgccgatcatcaccggcgctcgctccgtcccggtgagcggggagttcccgatatcgaacagccatggccatggcgagttctcctcttccctgcacaagcgcatccacccctacccggtgtctgagccagggagtgcaaagtgggacgagaagaaagaggtgagctggaaggagaggatggacgactggaaatccaagcagggcatcgtcgccggcggcgcccccgatcccgacgactacgacgccgacgtcccactgaacgacgaggcgaggcagccgctgtcgaggaaggtgtcgatcgcgtcgagcaaggtgaacccgtaccggatggtgatcatcctccgtctcgtggtgctcggcttcttcctccggtaccgcatcctccacccggtgcccgacgccatcccgctgtggctcacctccatcatctgcgagatctggttcgccgtgtcgtggatcctcgaccagttccccaagtggtacccgatcgacagggagacctacctcgaccgcctctccctccgctacgagcgcgagggggagccgtcgctgctgtcggcggtggacctgttcgtcagcacggtggatccgctcaaggagccgccgctggtcacggccaacaccgtgctgtccatcctcgccgtcgactaccccgtcgacaaggtgtcctgctacgtctccgacgacggcgcgtccatgctcacgttcgagtcgctgtcggagacggcggagttcgcccgcaagtgggtgccattctgcaagaagttcagcatcgagccccgcgccccggagttctacttctcccagaaggtcgactacctcaaggacaaggtccatcccaacttcgtccaggagcgccgcgccatgaagagagagtacgaggagttcaaggtgaggatcaacgcgctggtggcgaaggcgcagaaggtgccggcggaagggtggatcatgaaggacgggacgccatggccggggaacaacacccgcgaccacccgggcatgatccaggtgttcctcggccacagcggcggccacgacaccgagggcaacgagctcccccgcctcgtctacgtctcccgtgagaagcgccccggcttccagcaccacaagaaggccggcgccatgaacgccctcattcgtgtgtcggccgtgctgacgaacgcgccgttcatgctcaacttggattgcgatcactacatcaacaacagcaaggccatcagggaggcgatgtgcttcctcatggatccgcaggtcggacggaaggtttgctacgtgcagttcccgcagaggttcgacggcatcgacgtccacgaccgatacgccaaccgcaacaccgtcttcttcgacatcaacatgaaggggcttgatgggatccagggcccggtgtacgtcgggacagggtgcgtgttcaggcggcaggcgctgtacggatacaacccacccaagggacccaagaggcccaagatggtgacctgcgactgctgcccttgcttcgggaggaagaagcggaagcacggcaaggacggcctcccggaggccgtcgccgccgacggcgggatggacagcgacaaggagatgctcatgtcgcagatgaacttcgagaagcggttcgggcagtcggcggcgttcgtgacgtcgacgctgatggaggaaggcggcgtcccgccgtcgtccagccccgccgcgctcctcaaggaggccatccatgtcatcagctgcggctacgaggacaagaccgactggggtctcgagctggggtggatctacgggtcgatcacggaggacatcctaacggggttcaagatgcactgccgcgggtggaggtcggtgtactgcatgccgaagagggcggcgttcaaggggtcagcgccgatcaacctatctgaccgtctcaaccaggtgctccggtgggcgctcggctccgtcgagatcttcttcagccggcacagcccgctcctctacggctacaagaacggcaacctcaagtggctcgagcgcttctcctacatcaacaccaccatctaccccttcacttctctccccctcctcgcctactgcaccctacccgccgtctgcctcctcaccggcaagttcatcatgcctccgattagcacgtttgcgagtttgttcttcatcgcgctcttcatctccatcttcgcgacgggcatcctggagatgaggtggagcggggtgagcatcgaggagtggtggaggaacgagcagttctgggtcatcggcggcgtgtcggcgcacctgttcgcggtggtgcagggcctgctcaaggtgctggccgggatcgacaccaacttcaccgtcacgtccaaggccaccggagacgaggacgacgagttcgcggagctctacgccttcaagtggaccaccctcctcatcccgcccaccacgctgctcatcctcaacatcatcggcgtcgtcgccggcgtctccgacgccatcaacaacggctccgaggcgtggggcccgctcttcgggaagctcttcttcgccttctgggtcatcgtccacctctaccccttcctcaaggggctcatggggaggcagaaccggacgcccaccattgttgtcatctggtccgtgctgctcgcctccatcttttccttgctctgggtcaggattgatcccttcaccatcaaggccaggggccctgacgtcaggcagtgcggcatcaactgctga</cdnaseq>|
AA = <aaseq>MEASAGLVAGSHNRNELVLIRGHEEPKPLRALSGQVCEICGDEV GRTVDGDLFVACNECGFPVCRPCYEYERREGTQNCPQCKTRYKRLKGSPRVPGDEDEE DIDDLEHEFNIDDEKQKQLQQDQDGMQNSHITEAMLHGKMSYGRGPDDGDGNSTPLPP IITGARSVPVSGEFPISNSHGHGEFSSSLHKRIHPYPVSEPGSAKWDEKKEVSWKERM DDWKSKQGIVAGGAPDPDDYDADVPLNDEARQPLSRKVSIASSKVNPYRMVIILRLVV LGFFLRYRILHPVPDAIPLWLTSIICEIWFAVSWILDQFPKWYPIDRETYLDRLSLRY EREGEPSLLSAVDLFVSTVDPLKEPPLVTANTVLSILAVDYPVDKVSCYVSDDGASML TFESLSETAEFARKWVPFCKKFSIEPRAPEFYFSQKVDYLKDKVHPNFVQERRAMKRE YEEFKVRINALVAKAQKVPAEGWIMKDGTPWPGNNTRDHPGMIQVFLGHSGGHDTEGN ELPRLVYVSREKRPGFQHHKKAGAMNALIRVSAVLTNAPFMLNLDCDHYINNSKAIRE AMCFLMDPQVGRKVCYVQFPQRFDGIDVHDRYANRNTVFFDINMKGLDGIQGPVYVGT GCVFRRQALYGYNPPKGPKRPKMVTCDCCPCFGRKKRKHGKDGLPEAVAADGGMDSDK EMLMSQMNFEKRFGQSAAFVTSTLMEEGGVPPSSSPAALLKEAIHVISCGYEDKTDWG LELGWIYGSITEDILTGFKMHCRGWRSVYCMPKRAAFKGSAPINLSDRLNQVLRWALG SVEIFFSRHSPLLYGYKNGNLKWLERFSYINTTIYPFTSLPLLAYCTLPAVCLLTGKF IMPPISTFASLFFIALFISIFATGILEMRWSGVSIEEWWRNEQFWVIGGVSAHLFAVV QGLLKVLAGIDTNFTVTSKATGDEDDEFAELYAFKWTTLLIPPTTLLILNIIGVVAGV SDAINNGSEAWGPLFGKLFFAFWVIVHLYPFLKGLMGRQNRTPTIVVIWSVLLASIFS LLWVRIDPFTIKARGPDVRQCGINC</aaseq>|
DNA = <dnaseqindica>94..364#470..705#801..897#989..1115#1202..1814#1904..2167#2250..2462#2585..2792#2884..3080#3162..3515#3619..4206#agcgatcgatcgcccttcctcctcctcctctcctccttcctgcgtcgcctccctgatcagccgcagctcgttgccggccgttgttgcgcggccatggaggcgagcgccgggctggtggccgggtcgcacaaccggaacgagctggtgctgatccgggggcacgaggagcccaagccgctgcgggcgctgagcgggcaggtgtgcgagatatgcggcgacgaggtcggccgcaccgtcgacggcgacctcttcgtcgcctgcaacgagtgcggcttcccggtgtgccgcccctgctacgagtacgagcgccgcgagggcacccagaactgcccccagtgcaagacccgctacaagcgcctcaagggtaaaaaaacacactcacatcacgctacgcccttgataccgcgatcgcgaggtttccgttgattgatctctaatggcgatggtggtggtggtggttttgttacagggagcccgagggtgcccggggacgaggacgaggaggacattgacgacctggagcacgagttcaacatcgacgacgagaagcagaagcagctgcagcaggatcaggatggcatgcagaacagccacatcaccgaggcgatgctgcacggcaagatgagctacgggaggggccccgacgacggcgacggcaacagcaccccgctcccgccgatcatcaccggcgctcgctccgtcccggtacacacaacacacctcaccccactcacaccaaattctctccctcttctcacctcaacatgttcacgaaatgttctttgtaaaaaaaaattcaggtgagcggggagttcccgatatcgaacagccatggccatggcgagttctcctcttccctgcacaagcgcatccacccctacccggtgtctgagccaggtagtataacgaattcactacactaattaatcaatgttcttgatgaacacacattgatcatctcaatcatctaccgatgaattctgaccagggagtgcaaagtgggacgagaagaaagaggtgagctggaaggagaggatggacgactggaaatccaagcagggcatcgtcgccggcggcgcccccgatcccgacgactacgacgccgacgtcccactgtacgcaatcctcagctgagcagcttacactgatcatgcatgacaactagtatattgatcatgcctgaactctctgtggcttgcaggaacgacgaggcgaggcagccgctgtcgaggaaggtgtcgatcgcgtcgagcaaggtgaacccgtaccggatggtgatcatcctccgtctcgtggtgctcggcttcttcctccggtaccgcatcctccacccggtgcccgacgccatcccgctgtggctcacctccatcatctgcgagatctggttcgccgtgtcgtggatcctcgaccagttccccaagtggtacccgatcgacagggagacctacctcgaccgcctctccctccgctacgagcgcgagggggagccgtcgctgctgtcggcggtggacctgttcgtcagcacggtggatccgctcaaggagccgccgctggtcacggccaacaccgtgctgtccatcctcgccgtcgactaccccgtcgacaaggtgtcctgctacgtctccgacgacggcgcgtccatgctcacgttcgagtcgctgtcggagacggcggagttcgcccgcaagtgggtgccattctgcaagaagttcagcatcgagccccgcgccccggagttctacttctcccagaaggtcgactacctcaaggacaaggtccatcccaacttcgtccaggagcgccgcgccatgaaggtaatatcgatcgccatcgtcattgctgattctgtaggagcttgacgaagagctaactgttgtgggggcattggcatttgatcgagcagagagagtacgaggagttcaaggtgaggatcaacgcgctggtggcgaaggcgcagaaggtgccggcggaagggtggatcatgaaggacgggacgccatggccggggaacaacacccgcgaccacccgggcatgatccaggtgttcctcggccacagcggcggccacgacaccgagggcaacgagctcccccgcctcgtctacgtctcccgtgagaagcgccccggcttccagcaccacaagaaggccggcgccatgaacgccctcgtacgccacgccaccacatccttcatcttcaaaacaacacaactctgctcgatttgatcgaaatttctggtttgcttggcagattcgtgtgtcggccgtgctgacgaacgcgccgttcatgctcaacttggattgcgatcactacatcaacaacagcaaggccatcagggaggcgatgtgcttcctcatggatccgcaggtcggacggaaggtttgctacgtgcagttcccgcagaggttcgacggcatcgacgtccacgaccgatacgccaaccgcaacaccgtcttcttcgacgtgagctctctcccacacaaactagatgaatatatacaatcttgaaaaatccagagcaaatcacgatcgatcgagcaatgtgactgaaattttgtgtggtgcgttcttggtgagatcatcagatcaacatgaaggggcttgatgggatccagggcccggtgtacgtcgggacagggtgcgtgttcaggcggcaggcgctgtacggatacaacccacccaagggacccaagaggcccaagatggtgacctgcgactgctgcccttgcttcgggaggaagaagcggaagcacggcaaggacggcctcccggaggccgtcgccgccgacggcggtgagctcccaaattcagagctaagaagaaattatggtgtgagaagattttcagctgatggaataatgtaagtttgtgtgtggtggatcagggatggacagcgacaaggagatgctcatgtcgcagatgaacttcgagaagcggttcgggcagtcggcggcgttcgtgacgtcgacgctgatggaggaaggcggcgtcccgccgtcgtccagccccgccgcgctcctcaaggaggccatccatgtcatcagctgcggctacgaggacaagaccgactggggtctcgaggtaaccaccgttatcagacactaagccatattaccattgagtgagtttgtggtgtgaacgccatggatgtgtgtgatgcagctggggtggatctacgggtcgatcacggaggacatcctaacggggttcaagatgcactgccgcgggtggaggtcggtgtactgcatgccgaagagggcggcgttcaaggggtcagcgccgatcaacctatctgaccgtctcaaccaggtgctccggtgggcgctcggctccgtcgagatcttcttcagccggcacagcccgctcctctacggctacaagaacggcaacctcaagtggctcgagcgcttctcctacatcaacaccaccatctaccccttcacttctctccccctcctcgcctactgcaccctacccgccgtctgcctcctcaccggcaagttcatcatgcctccggtcagtcccatcccatcctgccatcaattgctcctcttcttccatggattttcccgccaaaactgaagtttcaaatgttctgaaccttttcttggtgatgcagattagcacgtttgcgagtttgttcttcatcgcgctcttcatctccatcttcgcgacgggcatcctggagatgaggtggagcggggtgagcatcgaggagtggtggaggaacgagcagttctgggtcatcggcggcgtgtcggcgcacctgttcgcggtggtgcagggcctgctcaaggtgctggccgggatcgacaccaacttcaccgtcacgtccaaggccaccggagacgaggacgacgagttcgcggagctctacgccttcaagtggaccaccctcctcatcccgcccaccacgctgctcatcctcaacatcatcggcgtcgtcgccggcgtctccgacgccatcaacaacggctccgaggcgtggggcccgctcttcgggaagctcttcttcgccttctgggtcatcgtccacctctaccccttcctcaaggggctcatggggaggcagaaccggacgcccaccattgttgtcatctggtccgtgctgctcgcctccatcttttccttgctctgggtcaggattgatcccttcaccatcaaggccaggggccctgacgtcaggcagtgcggcatcaactgctgagagagggcgtcagcgatttctgaagatttgtgcataggggggcagcaagaagatcgatttgtaaaagttttgtattgctcgtgttgagttcgtgctatgttcttctgtaatattttgggacccagaaatggtttaaacttttgaagagggaatggacagatggccatatttgaatgttaagcaacaagggctggtattctcactccatgtttgttagattttttgcagctgtgttctcattgctcctactagggatatatgggtaatttggaccaaaccatgggcattaatgtagcttaaaagtatatgaatcgatttggtcaagggctgaagtaatattcctacaaggaatatattcagattgcagc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001069742.1 RefSeq:Os09g0422500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 9]]
[[Category:Chromosome 9]]

Haoyajing cathy: Created page with "This Magnaporthe grisea resistance-sh was first discovered by Japanese scholars Imbe and Matsumoto in 1985. It's well known for its moderate resistance for Kyu77-07A in Shin..."

This Magnaporthe grisea resistance-sh was first discovered by Japanese scholars Imbe and Matsumoto in 1985. It's well known for its moderate resistance for Kyu77-07A in Shin-2.

==Annotated Information==
===Function===
R gene-mediated resistance is one of the most effective mechanisms of immunity against pathogens in plants. To date some components that regulate the primary steps of plant immunity have been isolated, however, the molecular dissection of defense signaling downstream of the R proteins remains to be completed. In addition, R genes are known to be highly variable, however, the molecular mechanisms responsible for this variability remain obscure.To identify novel factors required for R gene-mediated resistance in rice, we used rice insertional mutant lines, induced by the endogenous retrotransposon Tos17, in a forward screen involving the rice blast fungus Magnaporthe oryzae. We inoculated 41,119 mutant lines with the fungus using a high throughput procedure, and identified 86 mutant lines with diminished resistance. A genome analysis revealed that 72 of the 86 lines contained mutations in a gene encoding a nucleotide binding site (NB) and leucine rich repeat (LRR) domain-containing (NLR) protein. A genetic complementation analysis and a pathogenesis assay demonstrated that this NLR gene encodes Pish, which confers resistance against races of M. oryzae containing avrPish. The other 14 lines have intact copies of the Pish gene, suggesting that they may contain mutations in the signaling components downstream of Pish. The genome analysis indicated that Pish and its neighboring three NLR genes are high similar to one another and are tandemly located. An in silico analysis of a Tos17 flanking sequence database revealed that this region is a "hot spot" for insertion. Intriguingly, the insertion sites are not distributed evenly among these four NLR genes, despite their similarity at the sequence and expression levels.
Gene pyramiding is considered one of the most effective strategies for achieving durable resistance against blast disease (Magnaporthe oryzae B. Couch) in rice (Oryza sativa L.), although few studies have evaluated the combining effect of the resistance genes. We report the development of pyramided lines with two major blast resistance genes, Pish and Pib, and the evaluation of the combining effect of them. The two genes pyramided lines were selected from the progenies of a cross between one near isogenic line (NIL), which harbours Pish, and another NIL, which harbours Pib, in the genetic background of blast susceptible variety, CO 39. The presence of the resistance genes was confirmed by DNA markers linked to them. To obtain DNA markers for Pish, we genetically mapped the Pish locus. We confirmed the additive effect of Pish and Pib in the pyramided lines by their reaction patterns to blast isolates, suggesting the potential availabilities of the combinations of these genes. In addition, we provide DNA markers linked to Pish for marker aided selection in rice blast resistance breeding.
A gene analysis to identify the genes accounting for the moderate resistance of the rice variety Shin 2 and some other varieties to the blast fungus strain Kyu 77-07A was The differential varieties, Aichi Asahi (Pi-a) and carried out in the present study. Yashiro-mochi (Pi-ta), were found to be susceptible to the strain, while Ishikari Shirol*.e (Pi-i), Kanto 51 (Pi-k), Tsuyuake (Pi-k'!b), Fukunishiki (Pi-z) and Toride I (Pi-zt) were resistant, and Shin 2 (Pi-ks) and Pi N0.4 (Pi-ta2) moderately resistant. Therefore, Kyu 77-07A was classified as one of the strains belonging to race 102. Howver, the variety Reiho with the resistance gene Pi-ta2 was susceptible to this strain. Based on the knovn facts mentioned above, the objectives 0L the present study were' to determinA- whether Pi-ks is the gene responsible for the moderate resistance of Shin 2' to Kyu 77-07A along with the reason for the different reactions to Kyu 77-07A betweerL Pi N0.4 and Reiho which both have the same resistance gene Pi-ta2. For the gene analysis, the F2 or F3 plants of the crosses were inoculated with Kyu 77-07A and three other fungus strains using the spraying rnethod when the plants were at the four- to five-1eaf stage. Two varieties, Mineyutaka. and Saikai 155 belonging to the Shin 2 type varieties, were used as the representative parents of the varieties susceptible to Kyu 77-07A for the crosses.
===Expression===
Quantitative real-time RT-PCR analysis was carried out to investigate the expression pattern of Pish after infection with various M. oryzae races. The analysis revealed that there were no distinguishable alterations in Pish expression levels at different time points after inoculation with either incompatible or compatible races of the fungus, or after mock treatment. This result is consistent with previous reports that other Pi genes were constitutively expressed and not induced by pathogen challenge [10,11,13-15]. It is likely that most Pi genes are expressed before pathogen invasion and are post-transcriptionally regulated for activation of the signal transduction pathways leading to resistance responses.

===Evolution===
Classical genetics and genome analysis have demonstrated that R genes tend to be clustered. The 55-kb region containing the Pish locus contains three other NBS-LRR genes, all oriented in the same direction. Among the proteins encoded by these genes, Npi37-1 exhibits little similarity to the other three, while Npi37-3 shows high similarity to Npi37-2 (91%) and Pish (98%). Intriguingly, the N-terminal half of Npi37-3 is identical to that of Pish and the other half is identical to that of Npi37-2 . These data suggest that at first the ancestral NBS-LRR gene was duplicated to produce Npi37-1 and Npi37-2-pre. This would be followed by a second duplication event in which Npi37-2-pre was duplicated to produce Npi37-2 and Pish. More recently, a crossover and/or duplication presumably occurred between Npi37-2 and Pish, resulting in Npi37-2, Npi37-3, and Pish. Npi37-1 and Pish are identical between the cultivars NB and St. No.1, suggesting that the two paralogs, Npi37-2 and Npi37-3, probably mutated independently in NB and/or St. No.1 after the duplication events described above.

How did these frequent gene duplications in the Pish region occur? Although R gene loci in general tend to be duplicated, the mechanism for this duplication remains obscure. Gene duplication is sometimes caused by the misrepair of chromosomal double-strand breaks (DSBs), which arise spontaneously during the life of a cell. One possible inducer of DSBs is the endonuclease activity encoded by TEs. In Drosphila melanogaster, it has been reported that DSBs are important triggers of segmental duplication (SD), and the distribution of SDs correlates positively with that of TEs. Here we showed that the Pish locus is one of the hot spots for Tos17 insertionsc. Therefore, the Pish locus was presumably attacked frequently by the Tos17 endonuclease, resulted in DSBs, which might have caused gene duplication. In addition to this effect of endonuclease activity, the insertion of TEs is thought to be important for the molecular evolution of R genes. Actually, many types of TEs have been identified in R gene clusters. Recently, Hayashi and Yoshida reported that the insertion of a retrotransposon Renovator in the promoter region of the blast R gene Pit promoted its expression and reactivation, demonstrating that the insertion of TEs has contributed to R gene evolution.

However, the disease resistance genes annotated in the report were predicted from their DNA sequence similarity with sequences encoding NBS and/or LRR domains, therefore there was no direct evidence indicating they truly function as R genes or components of defense signaling. Here, we demonstrated that the functional R gene Pish is actually a hot spot of Tos17 insertion in the NB genome, and is the preferred target site among four highly conserved and closely linked NBS-LRR genes, even though the genes are highly similar at both the nucleotide sequence and expression levels. These results suggest that Tos17 inserts most frequently in functional genes within hot spot regions. A search of the FST database indicates that not all NBS-LRR gene loci are hot spots for Tos17 insertion. It is possible that the NBS-LRR gene loci that are hot spots for TE insertions are functional R genes that have not yet been identified. Thus, it may be possible to predict novel functional R genes in the FST database by looking for regions that are hot spots for TE insertions. This possibility should be assessed in the future.

The molecular mechanisms that determine TE integration site specificity in plants are still unknown. Studies of the Ty retrotransposons of yeast have revealed that interactions with bound chromosomal proteins can tether the Ty integration machinery to chromosomes and thereby direct integration to nearby sites . The human immunodeficiency virus (HIV) integrates preferentially into actively transcribed genes at sites with transcription-associated histone modifications. Therefore, it is possible that the insertion of Tos17 is regulated by chromatin structure or through interaction with chromatin binding proteins, rather than being controlled directly by the structures or expression levels of the targeted sequences.

===mutation===
To confirm whether the null mutation of Pish(t) caused disruption of the resistance mediated by Pish, we screened for allelic mutants among the 86 selected mutants from our screening. As expected, most of the mutants (72 of the 86 lines) had mutations caused by Tos17 insertions, deletions, or an unknown insertion in this locus. Because these mutant lines were produced by tissue culture and are derived from a relatively small number of induced calli, some of these mutations are shared in multiple independently regenerated plants. By considering which calli the mutants were derived from and by examining each mutation pattern (i.e., the positions of Tos17 insertions or the deletion sizes in those mutants), we determined that there are 56 independent mutant alleles at the Pish(t) locus among the 72 mutant lines. Of these, 46 alleles were caused by Tos17 insertion. The direction of Tos17 was not always the same and the insertion sites were dispersed evenly at this locus, suggesting that the insertion site within the locus was random rather than depending on specific DNA sequences. Nine independent deletion mutations were detected among thirteen lines. The deletion sizes were diverse, ranging from 24 bp to over 50 kb. One allele in the ND2032/ND2105/ND2452/ND2562 lines contained an unidentified insert of about 2.5-kb. In these mutants, the transcription of Pish(t) was barely detectable or not detected at all.
On the other hand, 14 of the 86 lines with diminished Pish-mediated resistance contained neither mutations, insertions, nor deletion in this locus. In these mutants, the expression of Pish(t) was no different from in the wild type plants. Therefore, we concluded that they are not pish(t) mutants and designated them ttm (tissue-culture triggered mutation). Although we cannot exclude the possibility that some of the ttm mutants have mutations in unknown R genes that correspond to the blast isolate carrying additional avr genes other than avrPish, others are likely to have mutations in components required for activation of Pish-mediated disease resistance.
To examine whether Pish(t) confer race-specific resistance, we transformed KM plants with an empty vector control and a construct containing the Pish(t) cDNA under the control of the cauliflower mosaic virus 35 S promoter. We obtained more than five independent transgenic lines for each construct, and used the T1 and T2 generations for the following analyses. Transgenic plants containing the Pish(t) construct were as healthy as KM plants transformed with the empty vector. When infected with a rice blast isolate containing avrPish, three independent transgenic lines expressing Pish(t) exhibited a resistance phenotype, whereas the lines containing the empty vector were susceptible. Thus, expression of the Pish(t) cDNA conferred Pish-mediated resistance on KM. To determine the resistance spectrum of Pish(t), the transgenic lines were inoculated with seven additional rice blast isolates. As expected, the transgenic KM plants containing Pish(t) exhibited the same pattern of resistance specificity as the donor cultivar NB. Thus, we concluded that Pish(t) is the Pish gene.

==Labs working on this gene==
1.Plant Disease Resistance Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan
2.Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Ibaraki, 305-8602, Japan
==References==
Please input cited references here.
1. Y. Koide;A. Kawasaki;M. J. Telebanco-Yanoria;A. Hairmansis;N. T. M. Nguyet;J. Bigirimana;D. Fujita;N. Kobayashi;Y. Fukuta
Development of pyramided lines with two resistance genes, Pish and Pib, for blast disease (Magnaporthe oryzae B. Couch) in rice (Oryza sativa L.)
Plant Breeding, 2010, 129(6): 670-675
2. Akira Takahashi;Nagao Hayashi;Akio Miyao;Hirohiko Hirochika
Unique features of the rice blast resistance Pish locus revealed by large scale retrotransposon-tagging
BMC Plant Biology, 2010, 10: 175
3. Tokio IMBE;Shohei MATSUMOTO
Inheritance of Resrstance of Rice Vanetles to the Blast Fungus Strains Virulent to the Variety "Reiho"
Japanese Journal of Breeding, 1985, 35(0): 332-339