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Du1

2014-06-05T03:07:56Z

Yangws: In the endosperm of japonica rice, du-1 and du-2 mutations cause the reduction of amylose contents.

==Annotated Information==
===Introduce===
''' The Wx gene encodes AD glucose starch glycosyl transferase and controls amylase synthesis in the endosperm of the grass family. Amylose accumulated in the rice endosperm is mainly regulated by the amount of Wx gene products. Amylose content is an important factor affecting the eating quality of rice, which varies markedly with countries and cultures. In rice, two Wx alleles, Wxa and Wxb, which regulate the amylose content widely. The endosperms homozygous for Wxa produce 10-fold more Wx gene products than those for Wxb, resulting in higher amylose content in the former. Wxb predominates in the Japonica type while Wxa in the India type and its relatives.
The importance of the amylose content in grain quality requires that the allelic states in parents be examined before hybridization and that unfavorable alleles in early generations after hybridization be eliminated. However, the level of Wx gene expression and amylose synthesis is also controlled by other genes controlling the amylose content. Especially, du genes are known to reduce the level of the Wx products as well as amylose in eh endosperm.

===Location===
Eight dull mutants that lower the amylose content of rice endosperm as well as waxy mutant and a cultivar with common grains were crossed in a dialyze manner. The amylose content of F 1 and F 2 seeds was determined on the basis of single grain analysis. It was concluded that the low amylose content of dull mutants is under monogenic recessive control. Alleles for low amylose content are located at five loci designated as du-1, du-2, du-3, du-4 and du-5. These loci are independent of wx locus located on chromosome 6. The five du loci have an additive effect in lowering the amylose content. Two loci, du-1 and du-4, were found to be located on chromosomes 7 and 4, respectively.

===Function===
The Wx gene expression plays a major role in determining the amylose content in the rice endosperm, although Wx alleles and their trans-acting genes like du genes are also known to genetically control the amylose content.
1. In endosperm, du-1 and du-2 reduce the amount of spliced Wxb transcripts. They affect either the splicing of intron 1 or other post-transcriptional steps in processing of the the Wxb transcripts.
2. Both du-1 and du-2 mutations cause inefficient splicing of the mutant Wxb transcripts that have three new weak 5′ splice sites due to a mutation at the 5′ splice site of intron 1, but not of the non-mutatedWxa transcripts.
3. Both du-1 and du-2 have differential effects on Wxb splicing in endosperm and pollen. Results suggest that neither du-1 nor du-2 encodes general factors required for pre-mRNA splicing.

===Experimental procedures===
1. Plant material
A japonica rice cultivar, Kinmaze, its mutant lines, du-1 (EM12) and du-2 (EM2) ( Yano et al. 1988 ), and other rice plants were all grown in a greenhouse under a light–dark cycle consisting of 14 h of light and 10 h of dark. Transgenic rice plants carrying the Wxa gus transgene were produced by electro oration of protoplasts isolated from embryogenic suspension cultures according to the method described previously. Transgenic plants of the R2 generation were crossed with dull mutants. F1 plants were selfed, and the F2 seeds and plants were screened for opaque dull endosperm and the presence of the Wxa-gus transgene by Southern blot analysis. F2 plants homozygous for both dull and Wxa-gus were selfed, and immature F3 seeds were analyzed by RNA gel blot analysis.
2. RNA gel blot analysis
Total RNA was prepared from immature seeds 15 days after pollination. RNA (10 μg) was separated by electrophoresis in 1% agarose gels containing formaldehyde, blotted and hybridized with a waxy cDNA probe that had been labeled with [α-32P] dCTP, using the Multiprime DNA labelling system (Amersham). The probes used were a 1.3 kb BglII fragment of Wx cDNA (exon 5 to exon 14), a 0.9 kb EcoRI fragment of RBE1 cDNA, a full length AGPP cDNA, a full length gus and the exon 2 of the rice actin 1. After the membrane was washed, an autoradiography was obtained by exposing the membrane to an X-ray film.
3. Competitive RT–PCR analysis of Wx transcripts
A competitor DNA was constructed by inserting a 61 bp DNA fragment into the BalI site in exon 3 of Wxa cDNA cloned into a pGEM-T vector. The competitor DNA was transcripted into RNA by the T7 promoter using Riboprobe Combination Systems. The competitor RNA was quantitated by absorbance at 260 nm. Total RNA was extracted from immature seeds and mature anther ( Chomczynski & Sacchi, 1987). Serial dilutions of the competitor RNA were co-amplified with 500 ng of total RNA using RNA PCR Kit (Takara) and LA Taq with GC Buffer (Takara). PCR cycle conditions followed by 25 or 30 cycles of 94°C for 30 sec, 68°C for 30 sec, 72°C for 1 min. To detect amplified products another two rounds of amplification were performed. The first amplification was 30 cycles with primers, 5′-ACCATTCCTTCAGTTCTTTG-3′, and 5′-TCCGTAGATCTTCTCACC-GG-3′, and the second cycle was 25 or 30 cycles with primers, 5′-CAGTTCTTTGTCTATCTCAAGACAC-3′ and 5′-CGTACCGAGGAG-AGATCACC-3′. The first primer pairs were used for amplification of endosperm RNA. After PCR reactions, amplified DNA was electrophoresed in 2% agarose gels and visualized by ethidium bromide staining. Quantitation of the amount of Wxb mRNA was performed as described previously.
4. RT–PCR analysis of other transcripts
RNA was amplified with 500 ng of total RNA using RNA PCR Kit (Takara). PCR amplifications were performed using RBE1-specific primer RB214 in exon 3, 5′-CATGGTGACTGTTGTGGAGG − 3′, and primer RB1500 in exon 6, 5′-CTATCAGGAATGGCCATTGC-3′, and AGPP-specific primer AG335 in exon 2, 5′-AACCGTCACCTGTCAA-GAGC-3′, and primer AG1203 in exon 7, 5′-TGAGTCCTCTATTATT-GCGCC-3′. PCR cycle conditions followed by 25 cycles of 94°C for 30 sec, 65°C for 30 sec, 72°C for 1 min. Amplified DNA was electrophoresed in 2% agarose gels and visualized by ethidium bromide staining.
5. Protein gel blot analysis
Proteins were extracted from mature seeds and anthers using a previously published protocol. For protein gel blot analysis, proteins were separated by 10% SDS-PAGE and transferred onto Immobilon polyvinylidene difluoride membranes (Millipore). After blocking with 1% BSA, the blots were probed by an antibody against rice WAXY protein ( Hirano & Sano, 1991). The immunoreactive protein was detected using an ECL Western blotting detection kit). An autoradiography was obtained by exposing the membrane to Hyperfilm-ECL. The detected signal was quantified by Densitograph (ATTO).

===Mutation===
Uniqueness of du-1 and du-2 mutations: our studies indicate that rice du-1 and du-2 mutations reduce the splicing efficiencies of intron 1 at two 5′ cryptic splice sites present in Wxb transcripts having + 1T rather than G. Furthermore, they seem to have no effect on the Wxa transcripts that have the normal 5′ splice site of intron 1. Moreover, these mutations do not appear to influence the splicing of transcripts derived from the three other genes examined. These findings suggest that du-1 and du-2 mutations may be uniquely uncovered due to the 5′ splice site mutation present in the Wxb allele of rice. However, our results do not exclude the possibility that gene products of du-1 and du-2 play roles in the splicing of other transcripts because they may quantitatively affect the splicing efficiencies of other transcripts or they may be redundant in particular tissues and/or developmental stages. In addition, dull mutants with low or intermediate amylose contents were not found in indica rice cultivars, most of which carry Wxa allele. These observations support the hypothesis that dull mutations only arise from plants having waxy genes with mutant 5′ splice sites. A mutant Drosophila lacking a splicing factor, SRp55, is lethal. In contrast, because the waxy gene is a non-essential gene, our studies may have identified mutations of factors affecting the splicing of waxy transcripts.
Mutant gene was allelic to du2 but the gene was designated as du2-2 as its phenotype was distinct. Nearisogenic lines (NILs) with different combinations of alleles at the Wx and du2-2 loci were then established and evaluated for the Wx gene expression. Waxy or chalky endosperms due to du2-2 were detected only in response to Wxb but not to Wxa, which may account for the fact that no du variants were detected in the Indica type.

===Discussion===
Two lines of evidence suggest that both du-1 and du-2 mutations affect the splicing of Wxb transcripts rather than post-transcriptional steps after splicing. First, they do not affect the processing of Wxa transcripts that are almost identical to Wxb transcripts including the 3′ UTR. Second, du-1 and du-2 show tissue-specific effects with respect to splice site selection and the abundance of spliced Wxb mRNA. These results are consistent with the current model on the regulation of pre-mRNA splicing based on studies in mammals and Drosophila as discussed below.
1. Possible roles of Du-1 and Du-2 proteins in splicing
A number of protein factors have been identified in vertebrates and yeast that are involved in the regulation of pre-mRNA splicing. For instance, SR proteins (reviewed in Manley & Tacke, 1996) are involved in constitutive and regulated splicing, are tissue-specifically regulated, and have been shown to bind purine-rich splicing enhancers present in exons. SR proteins have been identified in plants and have been shown to function as splicing factors in in vitro splicing assays with mammalian cell extracts. More recently, atSRp30, an Arabidopsis SR protein with a strong similarity to SF2/ASF, was shown to have an in vivo role in the regulation of pre-mRNA splicing in plants.
The selection of the 5′ splice site in early transcripts of SV40 has been shown to be regulated by SR proteins, and different SR proteins promote splicing at the two different 5′ splice sites. ASF/SF2, one of the best-characterized human SR proteins, has been shown to bind purine-rich exonic enhancers ( Tacke & Manley, 1995). Interestingly, examination of the base sequences of exon 1 of Wxb revealed the presence of an octamer, GGAAGAAC, which has a complete match with the consensus of the purine-rich exonic splicing enhancer ( Watakabe et al. 1993 ), RGAAGAAC, where R is any purine ( Tacke & Manley, 1995), at 17 bases upstream of the authentic splice site ( Isshiki et al. 1998 ). Based on these, it is an intriguing possibility that du-1 and du-2 code for splicing factor(s) such as SR proteins, binding to this putative splicing enhancer and stabilizing the splicing complex at two weak splice sites generated by mutation in Wxb pre-mRNA. Binding of these splicing factors may not be required for a strong wild-type 5′ splice site. Since multiple forms of SR proteins are also known in plants, our failure to identify the effects of du-1 and du-2 on the splicing of other transcripts in either endosperm or pollen may have resulted from their redundant functions.
Alternatively, du-1 and du-2 may encode more specific splicing regulators such as TRA or TRA 2, which are involved in sex-specific splicing of dsx transcripts inDrosophila ( Heinrichs & Baker, 1995; Tian & Maniatis, 1993), or hnRNP A1, which acts antagonistically to ASF/SF2 in the selection of the 5′ splice site.
The effects of du-1 and du-2 are tissue specific. Although both mutations equally affect splicing efficiencies at the two 5′ splice sites in endosperm, in pollen the du-1effect is greatly enhanced, whereas du-2 has little effect on splicing efficiencies. A tissue-specific difference in splicing efficiency was previously reported for a retrotransposon-induced mutant waxy gene of maize. The transcripts of wxG, in which a retrotransposon is inserted in intron 8, are 30-fold more efficiently spliced in pollen than in endosperm. This result suggests the presence of splicing factors that act differently in endosperm and pollen, and that the gene products of du-1 and du-2 may belong to these factors.
2. Use of the Wxbgene and dull mutations in studies of regulated splicing in plants
The study of splicing, particularly the identification of splicing regulators, is severely hampered by the lack of an in vitro system in plants. The Wxb system of rice described in this investigation may be a useful model system to study tissue-specific and regulated splicing in plants. Two weak 5′ splice sites were generated by natural mutations, and the selection of the splice site was tissue specifically regulated. Extragenic mutations influencing 5′ splice site selection in a tissue-specific fashion were available. A putative splicing enhancer which may interact with splicing regulators was present between the two weak 5′ splice sites of exon 1 ( Isshiki et al. 1998 ). The effects of variously modified cis-elements on splicing efficiency and splice site selection can be tested by transfection into rice protoplasts ( Isshiki et al. 1998 ). Having isolated three SR protein genes of rice, we are currently testing whether they may modulate splicing efficiency and selection of the two 5′ splice sites of the Wxb mRNA in rice protoplasts. Such studies should help us to understand the regulation of alternative splicing in the Wxb gene of rice.

==Labs working on this gene==
1. Ministry of Education, Science, Sports and Culture, Japan.
2. Laboratory of Plant Breeding, Faculty of Agriculture, Hokkaido University, Japan.
3. Biological Resources R&D Center, Fukui Prefectural University, Japan.
4. Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Japan.
5. Yokohama Research Center, Mitsubishi Chemical Co, Japan.
6. Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Japan.
7. Hokuriku National Agriculture Experiment Station, Japan.
8. Institute of Biological Chemistry, Washington State University, Washington.
9. Department of Biochemistry, Michigan State University, Michigan.
10. Department of Cell and Molecular Genetics, Scottish Crop Research Institute, UK.

==References==
1. M. Yano, K. Okuno, H. Satoh and T. Omura. Chromosomal location of genes conditioning low amylose content of endosperm starches in rice, Oryza sativa L. Theoretical and Applied Genetics, 1988, 76(2): 183-189.
2. Hikaru Satoh, Takeshi Omura. New Endosperm Mutations Induced by Chemical Mutagens in Rice Oryza sativa L. Japanese Journal of Breeding, 1981, 31(3): 316-326.
3. Le-Viet Dung, Ichiho Mikami, Etsuo Amano, etal. Study on the Response of dull endosperm 2-2, du2-2, to Two Wx Alleles in Rice. Breeding Science, 2000, 50(3): 215-219.
4. Masayuki Isshiki, Midori Nakajima, Hikaru Satoh, etal. Dull: rice mutants with tissue-specific effects on the splicing of the waxy pre-mRNA. The Plant Journal, 2000, 23(4): 451-460.
5. Anderson J, Hnilo J, Larson R, etal. The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme. J Biol Chem. 1989, 264(21):12238-12242.
6. Nakata P, Greene T, Anderson J, etal. Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits. Plant Mol Biol. 1991, 17(5):1089-1093.
7. Burton R, Johnson P, Beckles D, etal. Characterization of the genes encoding the cytosolic and plastidial forms of ADP-glucose pyrophosphorylase in wheat endosperm. Plant Physiol. 2002, 130(3):1464-75.

==Structured Information==
{{JaponicaGene|
GeneName = low amylose content mutant dul |
Description = Similar to CEL5=CELLULASE 5 (Fragment)|
Version = NM_001111411.1 GI: 162463770 GeneID: 541657|
Length = 6027 bp|
Definition = Zea mays dull endosperm1 (du1), mRNA.|
Source = Zea mays

ORGANISM Zea mays
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; PACMAD
clade; Panicoideae; Andropogoneae; Zea.
|
Chromosome = [[:category:Japonica Chromosome 7|Chromosome 7]]|
AP = Chromosome 7:1..6027|
CDS = 120..5144|
GCID = <gbrowseImage1>
name=NC_008401:762215..764081
source= Zea mays Chromosome07
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008401:762215..764081
source= Zea mays Chromosome07
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atgtgcagttggtcactctcgagccacactctcacttcgccggtgaggcaggcagcaatggagccaaagagcagcagctgcggcggcgccggcattcggctgcggctgctggtcgtgctccacctgctgctcttagttccgagctcggccatggcgttcaactacgccgacgcgctcgccaagtccatcatcttcttcgagggccagcgctccggcaagctcccgcccggcaaccgcatgccgtggcgcgccgactccggcctcaccgacggcgcccagtacaatgtggatttggtgggcgggtactacgacgccggcgacaacgtcaagttcggcctgcccatggcgttctcgacgacgatgctggcgtggagcgtgctcgacttcggcaagttcatgggcgccgagctgcccaacgcccgcgccgccgtgcgctggggcgccgactacctcctcaaggccgccaccgccacgcccggcgcgctctacgtccaggtcgccgaccccaaccaggaccaccgctgctgggagcgccccgaggacatggacacaccccgcagcgtctaccgcgtcaccgccgacaagccgggttccgacgtcgccggcgagacggccgccgcgctcgccgcgtcgtccatggtgttccgccgcgccgacccggcctactccgcgcgcctcctccacgccgcgacgcaggtgttcgacttcgccgaccggcaccgcgggtcgtacagcgactcgctggcgtcgtcggtgtgcccgttctactgctcctactcgggctaccacgacgagctcctgtggggggcgtcgtggctgcaccgcgcgtcgaggaacgcgtcgttcatgtcgtacgtggaggcgaacgggatgcagctcggcgccggggacgacgactactccttcagctgggacgacaagcgggtgggcaccaaggtgctcctcgccaagggcttcctccgcaaccgcctccatggcctcgagctctacaaggcgcactccgacagctacatctgctcgctggtgcccggcacggcgagcttccagtcgcggtacacccccggcggcctcctgtacagggaaggctccagcaacatgcagtacgtgacgacggcgacgttcctgatgctggcgtacgccaagtacctccggtcgagcggcgccaccgcgtcgtgcggcgacggcggcggcggagcgaggggggaggtgtcggcggcggagctggtggcggtggcgaagcggcaggtggactacatcctggggaagaacccggcggggatgtcgtacatggtggggttcgggtgcaggtacccgaggcgggcgcaccaccgcggcgcgtccatgccgtcggtgcgcgcccacccggggcggatctcctgcgacgccggcttcggctacctccactccggcgagcccaacccgaacgtgctcgtcggcgccgtcgtcggcgggccggacagccgcgacgcctttgccgacgaccgcggcaacttcgcgcagtcggagccggccacctacatcaacgcgccgctcgtcggcgcgctcgcctacttcgccggaaccaccaagtag</cdnaseq>|
AA = <aaseq> MEMVLRSQSPLCLRSGPVLIFRPTVAGGGGGTQSLLRTTRFARR
RVIRCVVASPGCPNRKSRTASPNVKVAAYSNYAPRLLVESSSKKSEHHDSSRHREETI
DTYNGLSGSDAAELTSNRDVEIEVDLQHISEEELPGKVSINASLGEMETVDEAEVEED KFEVDTSGIVLRNVAVREVDPKDEHNAKDVFVVDSSGTAPDNAAVEEVVDEAEVEEDM VDVDILGLDLNNATIEEIDLMEEALLENFDVDSPGNASSGRTYGGVDELGELPSTSVD CIAINGKRRSLKPKPLPIVRFQEQEQIVLSIVDEEGLIASSCEEGQPVVDYDKQEENS TAFDEQKQLTDDFPEEGISIVHFPEPNNDIVGSSKFLEQKQELDGSYKQDRSTTGLHE QDQSVVSSHGQDKSIVGVPQQIQYNDQSIAGSHRQDQSIAGAPEQIQSVAGYIKPNQS
IVGSCKQHELIIPEPKKIESIISYNEIDQSIVGSHKQDKSVVSVPEQIQSIVSHSKPN
QSTVDSYRQAESIIGVPEKVQSITSYDKLDQSIVGSLKQDEPIISVPEKIQSIVHYTK PNQSIVGLPKQQQSIVHIVEPKQSIDGFPKQDLSIVGISNEFQTKQLATVGTHDGLLM KGVEAKETSQKTEGDTLQATFNVDNLSQKQEGLTKEADEITIIEKINDEDLVMIEEQK SIAMNEEQTIVTEEDIPMAKVEIGIDKAKFLHLLSEEESSWDENEVGIIEADEQYEVD ETSMSTEQDIQESPNDDLDPQALWSMLQELAEKNYSLGNKLFTYPDVLKADSTIDLYF NRDLSAVANEPDVLIKGAFNGWKWRFFTEKLHKSELAGDWWCCKLYIPKQAYRMDFVF FNGHTVYENNNNNDFVIQIESTMDENLFEDFLAEEKQRELENLANEEAERRRQTDEQR RMEEERAADKADRVQAKVEVETKKNKLCNVLGLARAPVDNLWYIEPITTGQEATVRLY
YNINSRPLVHSTEIWMHGGYNNWIDGLSFAERLVHHHDKDCDWWFADVVVPERTYVLD
WVFADGPPGSARNYDNNGGHDFHATLPNNMTEEEYWMEEEQRIYTRLQQERREREEAI
KRKAERNAKMKAEMKEKTMRMFLVSQKHIVYTEPLEIHAGTTIDVLYNPSNTVLTGKP
EVWFRCSFNRWMYPGGVLPPQKMVQAENGSHLKATVYVPRDAYMMDFVFSESEEGGIY
DNRNGLDYHIPVFGSIAKEPPMHIVHIAVEMAPIAKVGGLGDVVTSLSRAVQDLGHNV
EVILPKYGCLNLSNVKNLQIHQSFSWGGSEINVWRGLVEGLCVYFLEPQNGMFGVGYV
YGRDDDRRFGFFCRSALEFLLQSGSSPNIIHCHDWSSAPVAWLHKENYAKSSLANARV
VFTIHNLEFGAHHIGKAMRYCDKATTVSNTYSKEVSGHGAIVPHLGKFYGILNGIDPD
IWDPYNDNFIPVHYTCENVVEGKRAAKRALQQKFGLQQIDVPVVGIVTRLTAQKGIHL
IKHAIHRTLERNGQVVLLGSAPDSRIQADFVNLANTLHGVNHGQVRLSLTYDEPLSHL
IYAGSDFILVPSIFEPCGLTQLVAMRYGTIPIVRKTGGLFDTVFDVDNDKERARDRGL
EPNGFSFDGADSNGVDYALNRAISAWFDARSWFHSLCKRVMEQDWSWNRPALDYIELY
RSASKL</aaseq>|
DNA = <dnaseqindica>74..358#444..1730# gaattccctagttcagagaaagaaagaagttgagaatgagaagcaagtgaggcgcgtttg ctgggaagtg gttcttgtga ggtttaggag ttcacccttcttttcttccccttctagaaatggagatggtcctacggtcgcagagccctctctgccttcggagtgggccggtgctcatttttcgaccaaccgtcgcgggcggaggagggggcactcagtctttgttgaggactaccagatttgcgagaagaagggtcattcgatgcgttgtagcaagtccaggttgtcctaataggaaatctaggacagcgtctcccaacgtaaaagtagctgcttatagcaactatgcgccaagactcctcgttgagtcaagctccaagaagagcgaacaccatgatagcagcagacaccgtgaagaaactattgatacatacaatgggctgtcaggttctgatgcagcgaattgacaagtaatagagatgtagaaattgaagtggatttgcagcacatttctgaggaggaattgccaggaaaagtatcgattaatgcatcattaggagaaatggaaacagtggatgaagctgaggtcgaggaggataagtttgaggtagatacctcaggaattgtattgcgcaatgttgcagttcgggaagtggatccaaaggatgaacataatgctaaagatgtatttgtggtagattcgtcaggaactgcaccagataatgctgcagtggaggaatggtagatgaagctgaggttgaagaggatatggttgatgtggatatcttgggacttgacttgaataatgcaacgatcgaggaaattgatttgatggaagaggctttactggagaacttcgacgtggattcaccaggcaatgcttctagtggtcgaacctatgggggtgtggatgagttgggtgagctgccttcaacatccgtggattgcatcgccattaacggaaaacgtagaagtttgaagcctaagcccttgccaattgtcaggttccaggaacaagaacagatagttttaagcatt gttgacgaag aagggttgat tgctagttca tgtgaagaag gccaaccggt ggtagattac gataagcaag aggaaaactctaccgctttc gatgaacaga agcaattaac tgatgatttc cctgaagaag gcatatctat agttcacttc cctgagccaa acaatgatattgttggatcc tcaaaattct tggagcaaaa acaagaattg gatggttcttataaacaaga tcgatcaacc actggattgc atgaacaagatcagtctgttgttagttcacacggacaagataaatcaattgttggtgtgcctcagcaaatccagtacaatgatcaatctattgctggttc tcatagacaa gatcaatcaa ttgccggtgc acctgagcaa atccaatccg ttgctggcta tataaaacca aatcaatcta ttgttggttc ttgtaaacaa catgaattga ttattcctga gcctaagaaa atcgaatcca tcatcagtta caatgaaata gatcaatcta ttgttggttc tcacaaacaa gacaaatctg ttgttagtgt gcctgagcaa atccaatccattgttagtca cagcaaacca aatcaatcta ctgttgattc ttatagacaa gctgaatcaa ttattggtgt gcctgagaaa gtccaatcca tcaccagtta cgataaacta gaccagtccattgttggttc tcttaaacaa gatgagccta ttattagcgt gcctgagaaa atccaatccattgtccatta cactaaacca aatcagtcta ttgttggctt gcccaaacaa caacaatcaa ttgttcatat cgttgaacca aaacagtcca tagatggttt ccctaaacaa gatctatcaatcgttggtat ctccaatgag tttcaaacaa agcaactggc tactgttggg actcatgatggattgcttat gaagggtgtg gaagctaagg agacatctca aaagactgaa ggggatacacttcaggcaac gttcaatgtc gacaacttgt cacagaaaca ggaaggctta actaaagaagcagacgagat aacaattatt gagaaaatca atgatgaaga ccttgtgatg attgaagaacagaaaagcat agccatgaat gaagaacaga cgattgttac cgaagaagac attccaatggctaaggttga gataggaatt gacaaggcca aatttttaca tctgctttct gaagaagagagttcatggga tgaaaatgaa gtgggaataa ttgaggctga tgaacagtat gaagtcgatg agacatctat gtccactgaa caagatatcc aggaatcacc taatgatgat ttggatccacaagcactatg gagtatgctt caagagcttg ctgaaaaaaa ttattcgctg ggaaacaagttgtttactta tccagatgta ttgaaagctg attcaacaat tgatctctat ttcaatcgtgatctatcagc tgtggccaat gagcctgatg tacttatcaa aggagcattc aatgggtgga agtggagatt tttcactgaa aaattgcaca agagcgagct ggcaggggac tggtggtgctgcaaactata cattcctaag caggcataca gaatggactt tgtgtttttt aacggacacacggtatatga aaataataac aataatgatt tcgtgataca aatagaaagc accatggatgaaaatttatt tgaggatttc ttggctgaag aaaagcaacg agaacttgag aaccttgcaaatgaggaagc tgaaaggagg agacaaactg atgagcagcg gcgaatggag gaagaaagggccgcagataa agctgacagg gtacaagcca aggttgaggt agagacgaag aagaataaattgtgcaatgt attgggttta gccagagctc ctgttgataa tttatggtac attgagcccatcacgactgg acaagaggct actgtcagat tgtattataa cataaactca agacctctagttcacagtac tgagatatgg atgcatggtg gctataacaa ttggattgat ggactctcttttgctgaaag gcttgttcat catcatgaca aagattgtga ttggtggttt gcagatgttgtcgtgcctga aagaacatat gtattggact gggtttttgc tgacggccca ccagggagtgcaaggaatta tgacaacaat ggaggacatg attttcatgc tacccttcca aataacatgactgaggaaga gtattggatg gaagaagaac aaaggatcta tacaaggctt caacaagagaggagggaaag ggaggaggct attaaaagga aggctgagag aaatgcaaaa atgaaagctgagatgaagga aaagactatg agaatgttcc tggtttctca gaaacacatt gtttacaccgaaccacttga aatacatgct ggaactacta ttgatgtgct ttataatcct tctaatacagttctaactgg aaagccagag gtttggtttc gatgttcctt taatcgttgg atgtatccaggtggggtgtt gccacctcag aagatggtac aagcagaaaa tggttcacac ctaaaagcaacagtttacgt tccacgagat gcctatatga tggacttcgt tttctcggag tcagaagaaggtggaattta tgataacaga aatgggttag actatcatat tcctgttttt gggtcaattgcaaaggaacc acctatgcac attgtccaca ttgctgttga gatggcacca atcgcaaagg ttggaggtct tggtgatgtt gtcactagtc tttcacgtgc tgtgcaagat ttaggacacaatgtggaggt tattcttcca aagtacggtt gcttgaatct aagcaatgtc aagaatctacaaatccatca gagtttttct tggggtggtt ctgaaataaa tgtgtggcgt ggactagtcgaaggcctttg tgtttacttc ctggaacctc aaaatgggat gtttggagtc ggatatgtattggcaggga cgatgaccgc cgatttggct tcttctgtcg ttctgctcta gagtttctcctccaaagtgg atcttctccg aacataatac attgccatga ttggtcaagt gctcctgttgcctggctaca caaggaaaac tacgcgaagt ctagcttggc aaacgcacgg gtggtattcaccatccacaa tcttgaattt ggagcgcatc atattggcaa agcaatgaga tattgtgataaagcaacaac tgtctctaat acatattcaa aggaagtgtc aggtcatggt gccatagttc ctcatcttgg gaaattctat ggcattctca atggaattga tccggatata tgggatccgt acaatgacaa ctttatcccg gtccactaca cttgtgagaa tgtggttgaa ggcaagagggctgctaagag ggcactgcag cagaagtttg ggttacagca aatcgatgtc cccgtcgtag gaatcgtcac tcgcctgaca gcccaaaagg ggatccacct gatcaagcat gcgattcaccgtacactcga acggaacgga caggtggttt tgcttggttc agcgccggac tctcgaatccaagctgattt tgtcaacctg gcgaatacgc tccacggcgt aaaccatggg caagtgaggctttccttgac ctacgacgag cctctctcgc atctgatata cgctggctct gacttcattctggtcccatc tatatttgag ccttgcggcc taactcagct cgtcgccatg cggtatggaa ccatcccgat tgtccgcaag actggagggc tcttcgacac tgtcttcgat gtggacaatgacaaggaacg agcccgagat cgaggccttg agcccaacgg gtttagcttt gacggagctg atagcaacgg tgttgactac gcgctgaaca gggcgatctc agcttggttc gatgcccggagctggttcca ctccctttgc aagagagtca tggagcagga ctggtcgtgg aaccgacctg ccctcgacta catcgagctc taccgttcag cgtccaaatt gtaataatcc aaacaacggccaatgtagtg tgttgtctgc aggtctcaga tgcagccatt cagcttttgc aggttcctgg gcattgctgt acagcctcct tgtctttagt tagctccatt ccccgaggag cacagtgcaattttttatcc tcagttatta tgcatagatt gtctcagtag aatgctttct tcgggcatgtatgtttgttt cctctgttgt tgaattctgg tgttaagtcg cgtataggaa tctacaggaaatgaaaaagt ccatttcctg cgtcaacctt ttagggctac catgcacatg agacctttcaagtgcaaaga atattaggac tagactacta gtatgtgaac tctatttttc caagagatttcaatttttcc aatgaaaaat aaactaattt ttcttggaaa aatggaaatc ccttggaaaa atggggttcc caaactagcc cgtagagtat agatcataga attggtctag tggttcctcg agagagaaaa aaacatagac ttttcttgtc atatgcttat ttaagtttat tttgtacaaa ctttgagaac cttcaaaaac accccaatgg ctggttaagt gaccagggaa ataaagaggatctataggga ggaatccccc gcctctctct cacagatgtt gcctagcacc ggccagcctc atccgtccag tggaattaag gttggttgcg acgacagccc atcaatggaa accaacctcg tgccccgtgc cgggatctac cttccttcct caccaccacg ccgatctcac cttccatagg agcttcctat gcactgttac ctattatagg tacatgacat tgtacatctt tgtatgaacttacatcaatg ccaaaaatcc ggaattc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001111411.1]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 7]]
[[Category:Chromosome 7]]

Du2

2014-06-04T15:06:47Z

Yangws: Created page with "==Annotated Information== ===Introduce=== '''The Wx gene expression plays a major role in determining the amylose content in the rice endosperm, although Wx alleles and their ..."

==Annotated Information==
===Introduce===
'''The Wx gene expression plays a major role in determining the amylose content in the rice endosperm, although Wx alleles and their trans-acting genes like du genes are also known to genetically control the amylose content. Experiments were conducted to determine whether genes inhibiting the Wx expression respond similary to two Wx alleles, Wxa and Wxb, which are predominantly distributed in cultivated. A recessive mutant with chalky endosperm and showing an independent inheritance of wx was used in this study. Complementation tests showed that the mutant gene was allelic to du2 but the gene was designated as du2-2 as its phenotype was distinct. Nearisogenic lines (NILs) with different combinations of alleles at the Wx and du2-2 loci were then established and evaluated for the Wx gene expression. Waxy or chalky endosperms due to du2-2 were detected only in response to Wxb but not to Wxa, which may account for the fact that no du variants were detected in the Indica type. The potential use of du2-2 for improving the grain quality of the Indica type was discussed.
===Function===
Both du-1 and du-2 have differential effects on Wx splicing in endosperm and pollen.Continuous planting of crops containing single disease resistance (R) genes imposes a strong selection for virulence in pathogen populations, often rendering the R gene ineffective. Increasing environmental temperatures may complicate R-gene-mediated disease control because high temperatures often promote disease development and reduceR gene effectiveness. Here, performance of one rice bacterial blight disease R gene was assessed in field and growth chamber studies to determine the influence of temperature on R gene effectiveness and durability.
Disease severity and virulence of Xanthomonas oryzae pv. oryzae (Xoo) populations were monitored in field plots planted to rice with and without the bacterial blight R geneXa7 over 11 yr. The performance of Xa7 was determined in high- and low-temperature regimes in growth chambers.
Rice with Xa7 exhibited less disease than lines without Xa7 over 11 yr, even though virulence of Xoo field populations increased. Xa7 restricted disease more effectively at high than at low temperatures. Other R genes were less effective at high temperatures.
===Expression===
In the rice genome, endo-1,4-b-D-glucanases form a multiple gene family including OsGLU3 which share high sequence similarity with KOR1 (2). OsGLU3is ubiquitously expressed in various tissues with strong expression in root tip, lateral root, and crown root primodia. OsGLU3contains four exons and three introns (Fig 2B). The putative OsGLU3 was predicted to contain a transmembrane domain, a cytosolic domain, and a catalytic domain ('''Supplemental Fig 3A'''). The mutation is located in the catalytic domain, which is highly conserved among the plant KOR1 homologs ('''Supplemental Fig 3B'''). qRT–PCR showed that the OsGLU3 is highly expressed in root tissue and has relatively lower expression in the other tissues. The OsGLU3–GUS expression was observed ubiquitously in the rice plants included in leaf veins, excoemums,and roots. the OsGLU3–GFP protein may reflect the native OsGLU3.OsGLU3 localizes in the plasma member and endosomes, and the export of OsGLU3 to the PM depends on vesicle transport. Phosphate starvation could induce root elongation inOsglu3-1.The phosphate starvation-induced primary root elongation and cellulose-content increase are abolished in Osglu3-2, which suggests that phosphate starvation-induced primary root elongation depends on the activity of OsGLU3.Suggesting that a single recessive gene was responsible for the mutant phenotype. Using 1 000 F2 mutant seedlings selected from the population, the mutation was mapped to a 56-kb region between S4-24837K and S4-24893K on chromosome 4. This region contains 14 open reading frames (ORFs), including a b-1,4-endoglucanase (OsGLU3, LOC_Os04g41970)(3).

===Evolution===
In rice genome, the putative membrane-anchored endo-b-1,4-D-glucanases were encoded by three genes: OsGLU1, OsGLU2, and OsGLU3.Recently,Libertiniet al.(2004) reported that 15 endoglucanase genes were present in rice genome.that these proteins could be classified into four main clusters. One cluster contained OsGLU4, OsGLU8, OsGLU12, OsGLU13,OsGLU14 and OsGLU15. Another cluster contained OsGLU1, OsGLU2, OsGLU3, KOR and CEL3. The third cluster contained OsGLU5,OsGLU6, OsGLU7, OsGLU9, OsGLU10 and OsGLU11.OsGLU1to OsGLU10 each gene had different numbers of introns and exons. All proteins of the OsGLU family contained the EGase domain. The
OsGLU1, OsGLU2 and OsGLU3 contained a predicted highly hydrophobic transmembrane motif in the N-terminal and belonged to the type II integral membrane protein anchored in the membrane. The results demonstrated thatOsGLU1, OsGLU2,OsGLU3 and OsGLU10 showed constitutive expression patterns in all the organs tested, and
the OsGLU4, OsGLU5, OsGLU6, OsGLU9were abundant in roots and developing flowers of plants. The other two genes OsGLU7 and OsGLU8 showed relatively higher expression in rachis and developing flowers. These different expression patterns indicated multiple functions of these genes in different processes of plant growth and development. Specific and combinational expression of these genes may be essential for the formation or function of a given organ（2）.
===Discussion===
The Osglu3-1 mutant has less cellulose in its roots and is defective in root cell elongation and division. However,theshoot development ofOsglu3-1seems ormal. In rice genome, the putative membrane-anchored endo-b-1,4-D-glucanases were encoded by three genes: OsGLU1, OsGLU2, and OsGLU3. Although all of them are
expressed ubiquitously in the rice plant,OsGLU1 showed high expression in shoot tissue whilstOsGLU3is highly expressed inroot tissue.This indicates that the different expression pattern of the gene members might explain the root elongation defect of Osglu3-1. Consistently with this,the mutation of OsGLU1 also resulted in a reduction in shoot cell growth(2). In our study, the exogenous glucose inhibits the primary root elongation in the WT, which might due to the osmotic stress or the glucose serving as a signal. However, it could completely restore the mutant phenotype ofOsglu3-1and partially restore the phenotype of Osglu3-2. There were two possible explanations for this phenomenon. One is that OsGLU3 might function in trimming sterol residues from nascent glucan primers. When glucose, the substrate of cellulose synthesis, is added, it leads to an increase in the glucan chain.The addition of the glucan chains together with the residual OsGLU3 enzymatic activity of the point mutation mutant could restore the phenotypic defects ofOsglu3-1. However, this explanation could not explain the partial complementation of the loss-of-function mutantOsglu3-2by the exogenous glucose. The other possibility is that OsGLU3 might hydrolyze the matrix polysaccharides or the links between matrix polysaccharides and, together with cell wall-loosening enzymes (such as expansins), create space for new synthesis cellulose.Exogenous glucose leads to an induction of cellulose synthesis,which needs more space(3).

==Labs working on this gene==
1.National Key Lab of Plant Genomics,People’s Republic of China.
2.Institute of Genetics and Developmental Biology, Chinese Academy of Sciences ,People’s Republic of China.
3.The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University,People’s Republic of China.
4.College of Science and Technology, Ningbo University, Ningbo, Zhejiang , China.
5.State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, People’s Republic of China.
6.Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou , People’s Republic of China.
7.Laboratoire de Biologie Cellulaire, Institut National de RechercheAgronomique
8.Universite´ de Rouen, CNRS UPRESA 6307, Faculte´ des Sciences
9.Centre de Physiologie Ve´ge´tale de l’Universite ´ Paul Sabatier, U.A.

==References==
1. Zhang J, Xu L, Wang F, Deng M, Yi K. Modulating the root elongation by phosphate/nitrogen starvation in an OsGLU3 dependant way in rice. Plant signaling & behavior. 2012;7(9):1144-5.
2. Zhou HL, He SJ, Cao YR, Chen T, Du BX, Chu CC, et al. OsGLU1, a putative membrane-bound endo-1,4-beta-D-glucanase from rice, affects plant internode elongation. Plant molecular biology. 2006;60(1):137-51.
3. Zhang JW, Xu L, Wu YR, Chen XA, Liu Y, Zhu SH, et al. OsGLU3, a putative membrane-bound endo-1,4-beta-glucanase, is required for root cell elongation and division in rice (Oryza sativa L.). Mol Plant. 2012;5(1):176-86.
4. Fre´de´ ric Nicol1, Isabelle His, Alain Jauneau, Samantha Vernhettes, Herve´ Canut, Herman Ho¨ fte. A plasma membrane-bound putative endo-1,4-betaD-glucanase is required for normal wall assembly and cell elongation inArabidopsis. The EMBO Journal. 1998;17(19):5563-76.

==Structured Information==
{{JaponicaGene|
GeneName = Os08g0114200|
Description = Similar to CEL5=CELLULASE 5 (Fragment)|
Version = NM_001067383.1 GI:115474502 GeneID:4344508|
Length = 1867 bp|
Definition = Oryza sativa Japonica Group Os08g0114200, 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 8|Chromosome 8]]|
AP = Chromosome 8:762215..764081|
CDS = 762288..762572,762658..763944|
GCID = <gbrowseImage1>
name=NC_008401:762215..764081
source=RiceChromosome08
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008401:762215..764081
source=RiceChromosome08
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atgtgcagttggtcactctcgagccacactctcacttcgccggtgaggcaggcagcaatggagccaaagagcagcagctgcggcggcgccggcattcggctgcggctgctggtcgtgctccacctgctgctcttagttccgagctcggccatggcgttcaactacgccgacgcgctcgccaagtccatcatcttcttcgagggccagcgctccggcaagctcccgcccggcaaccgcatgccgtggcgcgccgactccggcctcaccgacggcgcccagtacaatgtggatttggtgggcgggtactacgacgccggcgacaacgtcaagttcggcctgcccatggcgttctcgacgacgatgctggcgtggagcgtgctcgacttcggcaagttcatgggcgccgagctgcccaacgcccgcgccgccgtgcgctggggcgccgactacctcctcaaggccgccaccgccacgcccggcgcgctctacgtccaggtcgccgaccccaaccaggaccaccgctgctgggagcgccccgaggacatggacacaccccgcagcgtctaccgcgtcaccgccgacaagccgggttccgacgtcgccggcgagacggccgccgcgctcgccgcgtcgtccatggtgttccgccgcgccgacccggcctactccgcgcgcctcctccacgccgcgacgcaggtgttcgacttcgccgaccggcaccgcgggtcgtacagcgactcgctggcgtcgtcggtgtgcccgttctactgctcctactcgggctaccacgacgagctcctgtggggggcgtcgtggctgcaccgcgcgtcgaggaacgcgtcgttcatgtcgtacgtggaggcgaacgggatgcagctcggcgccggggacgacgactactccttcagctgggacgacaagcgggtgggcaccaaggtgctcctcgccaagggcttcctccgcaaccgcctccatggcctcgagctctacaaggcgcactccgacagctacatctgctcgctggtgcccggcacggcgagcttccagtcgcggtacacccccggcggcctcctgtacagggaaggctccagcaacatgcagtacgtgacgacggcgacgttcctgatgctggcgtacgccaagtacctccggtcgagcggcgccaccgcgtcgtgcggcgacggcggcggcggagcgaggggggaggtgtcggcggcggagctggtggcggtggcgaagcggcaggtggactacatcctggggaagaacccggcggggatgtcgtacatggtggggttcgggtgcaggtacccgaggcgggcgcaccaccgcggcgcgtccatgccgtcggtgcgcgcccacccggggcggatctcctgcgacgccggcttcggctacctccactccggcgagcccaacccgaacgtgctcgtcggcgccgtcgtcggcgggccggacagccgcgacgcctttgccgacgaccgcggcaacttcgcgcagtcggagccggccacctacatcaacgcgccgctcgtcggcgcgctcgcctacttcgccggaaccaccaagtag</cdnaseq>|
AA = <aaseq>MCSWSLSSHTLTSPVRQAAMEPKSSSCGGAGIRLRLLVVLHLLL LVPSSAMAFNYADALAKSIIFFEGQRSGKLPPGNRMPWRADSGLTDGAQYNVDLVGGY YDAGDNVKFGLPMAFSTTMLAWSVLDFGKFMGAELPNARAAVRWGADYLLKAATATPG ALYVQVADPNQDHRCWERPEDMDTPRSVYRVTADKPGSDVAGETAAALAASSMVFRRA DPAYSARLLHAATQVFDFADRHRGSYSDSLASSVCPFYCSYSGYHDELLWGASWLHRA SRNASFMSYVEANGMQLGAGDDDYSFSWDDKRVGTKVLLAKGFLRNRLHGLELYKAHS DSYICSLVPGTASFQSRYTPGGLLYREGSSNMQYVTTATFLMLAYAKYLRSSGATASC GDGGGGARGEVSAAELVAVAKRQVDYILGKNPAGMSYMVGFGCRYPRRAHHRGASMPS VRAHPGRISCDAGFGYLHSGEPNPNVLVGAVVGGPDSRDAFADDRGNFAQSEPATYIN APLVGALAYFAGTTK</aaseq>|
DNA = <dnaseqindica>74..358#444..1730#ACTCCCTCCTNTGCTGACAACAGATAACCACCTTTAACTGTAACTTTCCACAGCCTACCCCAGCCCTATA
AAGCTGCCNCTCTCCTATCTCCCTTCGCTGACTCTCTTTTCAGACTCAGCCCACTTGCACCCAAGTGAAT
TAACAGCCTTGTTGCTCACACAAAGCCTGTTTAGGTGGTCTTCTATATGGACATGCNTGACACTTGGTGC
CAAAATCTGGGCCAGGGGGACTCCTTCGTGAGACCGGCCCCCTGTCCTGGCCCTCATTCCGTGAAGAGAT
CCACCTGCGACCTCGGGTCCTCAGACCAGCCCAAGGAACATCTCACCAATTTCAAATCGGATCTCCTCGG
CTTAGTGGCTGAAGACTGATGCTGCCCGATCGCCTCAGAAGCCCCNTGGACCATCACAGATGCCGAGCTT
CGGGTAACTCTTACGGTGGAGGATTCCCAGCCATATGAAGACACCCTAGCTGGACGATCAGTCCTTGTCA
AAAGTCTGACCCCTCAAACTCTACAGCCTCAATGGACCAGACCCTACCCGGTCATTTATAGCACACCAAC
TGCCGTCCATCTGCAGGAACCTCTCCATTGGGTTCACCATTCCAGAATAAAGCCATGCCCATCAGACAGC
CAGCTTGATCTCTCCTCTTCCTCCTGGAAGCCACAAGATTAGGCCGAGAGCCGATCAGACAAACAACCTA
CAACCCTTAAGCTCCTGGCAGCGCCAAGCCAAGGCCATGCTTCCATGCAACACTCCTTCCAAATGGCCAT
CCCAGCATGCTTCCAAGCAGGCTTCATCCGTTCCTCTGGACCCTCATCTCTTAAGACCTGCCGCCTATAA
AAAGGATTATATCTTGAGACCCTATCCTCTAAAATTTTTTCCACACCCAAAACAAAAAATCTCTGGGTCA
AAAGTCTAAAACGCTTAGGCTGGCAACCATCAGATCCTTGCCCATGGTGTCCTCAAGCCTACTCTCATGA
AATGGACAACAGTACACGCATATGGGGCCAGTTCCACATATTTGGCAACCAGACCAGCATCCAGGACAAC
ACAAAGTATGTTGTTTGTTGTTAGAGGGCTTGGGACATTTCACTCTTTGCCAGCCTCAGCTTAATCCAGG
AGACAAAGATTATTTTCCTTATTATCTCTTCTGCATAGGATCTGCAATCAGAACTATTGAACTTCTCCAT
TCAGACCGCCACTCACACCTATGGGAAAAGGGTAATGTATCATCGGCTTAGCAACAGGGTATACTATTCG
TATGATGGAAAATGGGGACAAAAGGCTTTGGTACATCATAC</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001067383.1 RefSeq:Os08g0114200]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 8]]
[[Category:Chromosome 8]]