Os01g0841500
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Contents
Annotated Information
Over-expression of OsMYB3R2 (Os01g0841500) may significantly improve cold tolerance by mediating the alteration in cell cycle and ectopic expression of stress genes in rice.
Function
OsMYB3R-2 functions in both stress and developmental processes in rice (Oryza sativa). Transgenic plants overexpressing OsMYB3R-2 exhibited enhanced cold tolerance. Cold treatment greatly induced the expression of OsMYB3R-2, which encodes an active transcription factor. We show that OsMYB3R-2 specifically bound to a mitosis-specific activator cis-element, (T/C)C(T/C)AACGG(T/C)(T/C)A, a conserved sequence that was found in promoters of cyclin genes such as OsCycB1;1 and OsKNOLLE2. In addition, overexpression of OsMYB3R-2 in rice led to higher transcript levels of several G2/M phase-specific genes, including OsCycB1;1, OsCycB2;1, OsCycB2;2, and OsCDC20.1, than those in OsMYB3R-2 antisense lines or wild-type plants in response to cold treatment. Flow cytometry analysis revealed an increased cell mitotic index in overexpressed transgenic lines of OsMYB3R-2 after cold treatment. Furthermore, resistance to cold stress in the transgenic plants overexpressing OsCycB1;1 was also enhanced. The level of cellular free proline was increased in the overexpressed rice lines of OsMYB3R-2 and OsCycB1;1 transgenic plants compared with wild-type plants under the cold treatment. These results suggest that OsMYB3R-2 targets OsCycB1;1 and regulates the progress of the cell cycle during chilling stress. OsCPT1, which may be involved in the dehydration-responsive element-binding factor 1A pathway, showed the same transcription pattern in response to cold as did OsCycB1;1 in transgenic rice. Therefore, a cold resistance mechanism in rice could be mediated by regulating the cell cycle, which is controlled by key genes including OsMYB3R-2. Our previous studies have indicated that OsMYB3R-2 transgenic Arabidopsis plants are more resistant to freezing, drought, and salt stresses, leading us to speculate whether the regulation of the cell cycle is involved in the OsMYB3R-2-modulated stress response. Here, we show that OsMYB3R-2 is involved in regulating the responses to cold stress in rice. We demonstrate that OsMYB3R-2 functions as a MYB3R transcription factor targeting to OsCycB1;1, which is involved in the G2/M phase transition at low temperature. The transcript level of OsCPT1, a putative member of the dehydration-responsive element-binding factor 1 (DREB1)/CBF pathway, is also enhanced by OsMYB3R-2, accompanied by an increased Pro level. Our data indicate that in rice, OsMYB3R-2 may play an important role in the cold stress signaling pathway modulated by the cell cycle and a putative DREB/CBF pathway. Molecular Characteristics and Phenotypes of OsMYB3R-2 Transgenic Rice Transgenic lines of OsMYB3R-2 in rice were confirmed by hygromycin selection and GUS staining. The results of northern blot and real-time PCR showed that expression of OsMYB3R-2 was increased in the four independent overexpressing lines but decreased in the four antisense lines (Fig. 1, A and B). Southernblot analysis with a specific GUS probe showed diverse expression patterns in the four overexpressed lines as well as in the four antisense lines (Fig. 1, C and D). To examine the expression patterns of OsMYB3R-2 in vivo, transgenic rice lines were generated with a GUS expression construct driven by a putative OsMYB3R-2 promoter of 1,285 bp in length. GUS staining assay of T1 rice plants showed strong signals in nearly all tissues examined, including roots, internodes, nodes, leaf blades, lamina joints, sheaths, glumes of flower organs, and young embryos of immature seeds (Fig. 1E), suggesting that OsMYB3R-2 is a constitutively expressed gene. Transgenic rice seeds of either the OsMYB3R-2 overexpressors or the antisense lines did not differ from wild-type seeds in germination (Fig. 2). However the overexpressing plants showed growth retardation in comparison with wild-type plants under normal conditions (Fig. 2, B and C). The length of the root cell resulted in shorter roots in the overexpression transgenic lines (Supplemental Fig. S3). When wild-type plants reached the tetraphyllous stage, more than 80% of the OsMYB3R-2 overexpression plants were still at the trefoil stage (Fig. 2B). Growth retardation was observed in transgenic plants up to the heading stage.
Overexpression of OsMYB3R-2 Enhanced Tolerance to Chilling Stress in Rice To test the possible effect of OsMYB3R-2 expression on tolerance to chilling, the T2 transgenic and wildtype seedlings at the trefoil stage were exposed to reduced temperature (2℃) for various durations, followed by incubation at a normal growth condition in a greenhouse for 2 weeks. Fewer than 20% of the wild-type seedlings survived a cold treatment of 72 h, but none of them was able to resume growth when transferred to normal growth conditions. However, more than 50% of the OsMYB3R-2-overexpressing seedlings could survive and grow normally (Fig. 3, B and C). The survival ratio of antisense seedlings was less than that of the wild type. A time course of treatment showed drastic differences as the process was extended (Fig. 3D). The wild-type and transgenic seedlings showed no growth differences after chilling treatment for up to 48 h. In contrast, when the time of treatment was extended up to 60 h, more than 80% of the OsMYB3R-2-overexpressing plants grew normally, as compared with 55% of the wild-type seedlings and 45% of the antisense seedlings. Finally, after 84 h, neither wild-type nor antisense plants survived; in contrast, 20% of the overexpression lines were still healthy. Therefore,OsMYB3R-2 plays an important role in regulating tolerance against chilling stress in rice.
To investigate the functions of OsMYB3R-2 in DREB/CBF stress pathways, we tested the expression patterns of more than 10 genes by reverse transcription (RT)-PCR in wild-type and transgenic rice plants. One of the genes, OsCPT1, was activated by OsMYB3R-2 under cold stress, a deduced target gene of the DREB pathway with the DRE/CRT cis-elements (Fig. 4A). A DRE/CRT cis-element, CCGACCT, appeared in the upstream sequence (602–596 bp) of the OsCPT1 promoter. The transcription levels of rice DREB genes, including OsDREB1A, OsDREB2A, and OsCBF, did not show any changes in OsMYB3R-2-overexpressing transgenic lines under the cold conditions compared with the wild type. For the expression patterns of other cold-regulated (COR) genes such as OsCORTM1 and OsMAT1, which are the rice homologs of target genes of Arabidopsis DREBs (Dubouzet et al., 2003; Chen et al., 2008; Supplemental Fig. S1; Supplemental Table S1), there were no differences between the transgenic lines and the wild type. These data suggest that OsMYB3R-2 may regulate the plant response to cold stress through the deduced OsCPT1-involved DREB/CBF pathway in rice. Under normal growth conditions (25℃), the levels of cellular free Pro did not differ between wild-type and transgenic rice in the range of 112 to 118 mg fresh weight of material (Fig. 4B). In contrast, after cold treatment (2℃), the levels of free Pro in OsMYB3R-2-overexpressing transgenic rice increased substantially, with more than 300 mg g21 fresh weight compared with 188 mg g21 fresh weight in the wild-type plants. These results were similar to the alterations observed in other transgenic plants overexpressing the resistant genes, such as OsDREB1, OsCOIN, OsCIPK03, and OsCIPK12, which showed resistance to cold stress in rice (Ito et al., 2006; Liu et al., 2007; Xiang et al., 2007). Thus, cellular free Pro level is involved in enhanced resistance to cold regulated by OsMYB3R-2 via a putative DREB/CBF-CPT pathway in rice.
OsMYB3R-2 Protein Showed Transcription Activation A yeast GAL4 system was used to investigate the transcription activation of OsMYB3R-2 (Fig. 5A). OsMYB3R-2 mutants deleted in various domains were tested. The N and C termini of OsMYB3R-2 were truncated and the products were termed numbers 2 to 14, with the full-length protein termed number 1. Figure 5 shows a yeast growth analysis on screened medium with SD/-Trp/-Ade, SD/-Trp/-His, or SD/-Trp/-His/-Ade (see “Materials and Methods”) as well as in the galactosidase assay. Stronger blue signals corresponding to good growth of yeast on both media appeared in numbers 1, 2, 3, 9, 11, and 13 compared with the control empty vector and the remaining constructs. A common region among constructs was the region of 350 to 500 amino acids at the C terminus. These results suggest that the OsMYB3R-2 protein has transcriptional activation activity, and the core region with the activity was from 350 to 450 at theNterminus.
Bioinformatic analysis showed MSA-like sequences in the promoters of cyclin genes in rice (La et al., 2006). Two MSA-like sequences of OsCycB1;1 appeared between 2200 to 2400 bp upstream of the transcription start site. A fully conserved central core pentamer, AACGG, was found in the MSA-like elements. There is a 3-bp less conserved sequence at each side of the core motif. The MSA consensus sequence (T/C)C(T/ C)AACGG(T/C)(T/C)A is shown in Figure 6A (Ferreira et al., 1994; Day et al., 1996; Ito et al., 1997, 1998). It matches the consensus sequences of c-Myb and v-Myb binding sites (Howe and Watson, 1991; Grotewold et al., 1994; Ito et al., 1998), which suggests that the Myb transcription factors may bind the MSA motif. To test whether OsMYB3R-2 interacts with the MSA motif in the promoter of OsCycB1;1, electrophoretic mobility shift assay (EMSA) was carried out. EMSA showed that OsMYB3R-2 can specifically bind the OsCycB1;1 promoter of a 378-bp fragment in rice (Fig. 6, B and C). EMSA analysis of two MSA elements (RT1 and RT2) from the OsCycB1;1 promoter (Fig. 6, B and D) showed that the mobility of OsMYB3R-2 specifically shifted with the MSA elements from type B cyclin genes on the membrane map (Fig. 6E). Assay of the mutated RT1 sequence of the OsCycB1;1 promoter showed that any mutation could weaken the DNA-binding ability of OsMYB3R-2 protein (Fig. 6E), whereas the DNA-binding ability was abolished by base substitution of RT1mut5. Therefore, we concluded that the CCCAACGG sequence in the OsCycB1;1 promoter was recognized by OsMYB3R-2 protein.
We further examined the expression patterns of genes related to the cell cycle (Supplemental Table S2). Compared with the expression levels of types A and D cyclins, the expression levels of the type B cyclin genes OsCycB1;1, OsCycB2;1, OsCycB2;2, and OsCDC20.1 were suppressed by cold treatment in both the wild-type and antisense plants. In the OsMYB3R-2-overexpressed lines under cold treatment, expression patterns of those type B cyclin genes were the same as those at room temperature (25℃; Fig. 7A). To test whether the cold tolerance phenotype could be reproduced by overexpressing OsCycBs, transgenic rice overexpressing OsCycB1;1 as well as RNA interference (RNAi) lines (Supplemental Fig. S2) were tested for cold stress. The results showed that less than 58% of wild-type seedlings could survive after the treatment for 72 h, whereas more than 67% of overexpressed OsCycB1;1 seedlings could survive and grow normally (Fig. 7B). In contrast, less than 33% of OsCycB1;1-RNAi seedlings could resume growth under normal growth conditions. Our data showed that the overexpressing lines of OsCycB1;1 enhanced the tolerance to chilling stress compared with the wild type. This suggests that OsCycB1 is likely to be one of the downstream genes regulated by OsMYB3R-2 under chilling stress. To investigate whether there is any relationship between Pro level and resistance to cold stress in OsCycB1;1-overexpressing transgenic rice plants, the level of cellular free Pro was monitored. The results showed that under normal growth conditions (25℃), the levels of cellular free Pro were similar in both wildtype and OsCycB1;1 transgenic rice at a range of 120 to 124 mg g21 fresh weight of material (Fig. 7C). In contrast, after cold treatment (2℃), the level of free Pro in OsCycB1;1-overexpressing transgenic rice increased substantially, with 243 mg g21 fresh weight compared with 197 mg g21 fresh weight in the wild type and 185 mg g21 fresh weight in OsCycB1;1-RNAi plants. These results of the changed pattern for cellular free Pro were similar to those in OsMYB3R-2 transgenic plants. Taken together, the data suggested that OsCycB1;1 was directly regulated by OsMYB3R-2, which was involved in the tolerance to cold in rice.
Based on the results of the expression levels of cyclins, we monitored the mitotic index of OsMYB3R- 2-overexpressed lines under cold conditions. Mitotic index is defined as the ratio between the number of cells in mitosis and the total number of cells, which is used as a measure for the proliferation status of a cell population. Flow cytometry revealed that the DNA content of the OsMYB3R-2-overexpressed lines increased at 4℃ compared with the wild type under normal (28℃) and cold (4℃) conditions (Fig. 8, A–D). Thus, the overexpressing lines possessed more cells in the G2/M phase, especially under the cold conditions. Under the normal conditions, the overexpressing lines showed a higher mitotic index than wild-type and antisense lines. Under cold conditions, in contrast, the mitotic index in the overexpressing lines was markedly higher than that of the wild type, and the index of the antisense lines was notably lower than that of the wild type under cold stress (Fig. 8E). The decreased percentage of the mitotic index under cold stress compared with normal conditions (28℃) was 24.3%, 11.6% to 14.5%, and 33.5% to 38.6% in the wild type, the overexpressing lines, and the antisense lines, respectively. The changes in the mitotic index correlated to the expression pattern of OsMYB3R-2. Therefore, we conclude that OsMYB3R-2-overexpressing lines possess more cells at the G2/M cell cycle phase, which promoted an increased mitosis.
Expression
GUS staining shows expression pattern of OsMYB3R-2 in vivo in various tissues from the T1 generation of OsMYB3R-2 promoter::GUS transgenic rice. a, Root; b, young internode; c, mature internode; d, node; e, mature leaf; f, lamina joint; g, leaf sheath; h, flower; i, immature seed.
Evolution
A genome-wide analysis identified at least 155 and 197 MYB genes in rice and Arabidopsis, respectively. Gene structure analysis revealed that MYB family genes possess relatively more number of introns in the middle as compared with C- and N-terminal regions of the predicted genes. Intronless MYB-genes are highly conserved both in rice and Arabidopsis. MYB genes encoding R2R3 repeat MYB proteins retained conserved gene structure with three exons and two introns, whereas genes encoding R1R2R3 repeat containing proteins consist of six exons and five introns. The splicing pattern is similar among R1R2R3 MYB genes in Arabidopsis. In contrast, variation in splicing pattern was observed among R1R2R3 MYB members of rice. Consensus motif analysis of 1kb upstream region (5′ to translation initiation codon) of MYB gene ORFs led to the identification of conserved and over-represented cis-motifs in both rice and Arabidopsis. Real-time quantitative RT-PCR analysis showed that several members of MYBs are up-regulated by various abiotic stresses both in rice and Arabidopsis.
Knowledge Extension
The chilling tolerant LTH showed a different constitutive gene expression profile compared to the chilling-sensitive IR29. The dominant change in gene expression at low temperature was up-regulation in the chilling-tolerant genotype and down-regulation in the chilling sensitive genotype. Early responses to chilling stress common to both genotypes featured up-regulated genes related to transcription regulation and signal transduction, while functional categories of LR-chilling regulated genes were clearly diverse with a wide range of functional adaptations in two genotypes. Thirdly, at the end of the chilling treatments, there was quick and efficient reversion of gene expression in the chilling-tolerant LTH, while the chilling sensitive IR29 displayed considerably slower recovery capacity at the transcriptional level. Analysis of differentially- regulated TF genes and enriched cis-elements demonstrated that multiple regulatory pathways, including CBF and MYBS3regulons, are involved in chilling stress tolerance.
Labs working on this gene
Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences. Engineering Research Center for Plant Biotechnology and Germplasm Utilization, Ministry of Education, State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan, China. University.WuhanInstitute of Crop Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing China. IRRI-Korea Office, National Institute of Crop Science, Republic of Korea
References
Xu M R, Cruz C M V, Fu B Y, et al. Different patterns of gene expression in rice varieties undergoing a resistant or susceptible interaction with the bacterial leaf streak pathogen[J]. African Journal of Biotechnology, 2013, 10(65): 14419-14438. Zhang T, Zhao X, Wang W, et al. Comparative transcriptome profiling of chilling stress responsiveness in two contrasting rice genotypes[J]. PloS one, 2012, 7(8): e43274. Ma Q, Dai X, Xu Y, et al. Enhanced tolerance to chilling stress in OsMYB3R-2 transgenic rice is mediated by alteration in cell cycle and ectopic expression of stress genes[J]. Plant Physiology, 2009, 150(1): 244-256. Katiyar A, Smita S, Lenka S K, et al. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis[J]. BMC genomics, 2012, 13(1): 544.
Structured Information
| Gene Name |
Os01g0841500 |
|---|---|
| Description |
Similar to C-myb-like transcription factor (Fragment) |
| Version |
NM_001051302.1 GI:115440974 GeneID:4327399 |
| Length |
5656 bp |
| Definition |
Oryza sativa Japonica Group Os01g0841500, 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 | |
| Location |
Chromosome 1:37878633..37884288 |
| Sequence Coding Region |
37879090..37879259,37880370..37880421,37880776..37880833,37881239..37881333,37881419..37881522 |
| Expression | |
| Genome Context |
<gbrowseImage1> name=NC_008394:37878633..37884288 source=RiceChromosome01 preset=GeneLocation </gbrowseImage1> |
| Gene Structure |
<gbrowseImage2> name=NC_008394:37878633..37884288 source=RiceChromosome01 preset=GeneLocation </gbrowseImage2> |
| Coding Sequence |
<cdnaseq>atgggggccatggcgatggtggagcaggaggggtgcgtcgagaaccggcagcccctggcggcgtcgagctcgtcagtttccgacggaagcagctatggcggcggcggcggcgggctcgcgcagatgtcgccgccggtgtctagctcggcgaattccatctccggtcttaggcggacaagcgggccaataaggagagcaaagggtggctggacaccagaggaggacgagacattgaggaaggcagttgaggcttataagggtagaaactggaagaaaatagccgaatgttttccatacagaacagaggtacaatgcttgcatcgatggcaaaaggttcttaatcctgaacttatcaaaggtccttggactcaagaggaagatgatcaaattattgatcttgtaaagaagtatggaccaacaaaatggtctgtcatagctaaggcattacctggccgtatagggaagcaatgtcgagagaggtggcacaatcatctaaatccagaaataaggaaagatgcttggactactgaggaggaacaggcactcataaatgctcatcggatttatgggaataaatgggcagagatagcaaaagttcttcctggaaggactgataattctataaagaatcattggaatagttctctcagaaagaagcaagatatgtacaataccagcaataatatggtagttccgaaactgcttgttcatgataagttcaaggataaaccaaagctgatggctatggagggtcatcttgatttgaacaaggcacccattatcaattcaaaagaccaacctggaacagctcatcggtctaattgctcagggtttctatctcgttcctcattaccaactgctcagccacttacttcccgtgaggcatctgtagttgatggttccgctgtcactttagtggcgcaagctctggaaagtgattctgtccgtggtaagggcttggaaattgattctgttcatgagaaaggtcttgaggttaactcggcacctgatcataccgggaactcttggaccattcaactagaagctgcaccatccaaaggtgaagcagaattatctttgaagaatgaggcacgctctcttggtccactttgttaccagatccctaacatggaagatgtagtccctgtgagttcctcactcttttctgaccatcttactgggaaccatacatctgaacattgtggggatgatatattgtcacctgctggctgcactactccccctccaacaaagggaaaattaacaagccagcttagtgttgattcaattttgaagagtgctgctaacagttttccaggtactccttcaatattaaaaagaagaaaacgggataaatcaacacctgtttcagctagtgagatgaagatcagtggatcaaatactgatagattttacactcccatggggatggaacctgctacagctacaccagagtcattcaaaactacatcttttttgtcattgggttctcttgatggttctgtaaagagctttgatgtttcaccacaataccgggcaaggtcaaaaagaatggctctcacgaaaacagttgagaaacagctggatttttcatcagatggattagatacttgtggttctgagattctgaactcttcctgcaataattcccaaagcacactttctattaccgaagccccaaaactgaaagaaaaggaacatgctgttcaattggaaaatttaaccaagaactttgcgcatacaaccaatttggatgtaacctaa</cdnaseq> |
| Protein Sequence |
<aaseq>MGAMAMVEQEGCVENRQPLAASSSSVSDGSSYGGGGGGLAQMSP PVSSSANSISGLRRTSGPIRRAKGGWTPEEDETLRKAVEAYKGRNWKKIAECFPYRTE VQCLHRWQKVLNPELIKGPWTQEEDDQIIDLVKKYGPTKWSVIAKALPGRIGKQCRER WHNHLNPEIRKDAWTTEEEQALINAHRIYGNKWAEIAKVLPGRTDNSIKNHWNSSLRK KQDMYNTSNNMVVPKLLVHDKFKDKPKLMAMEGHLDLNKAPIINSKDQPGTAHRSNCS GFLSRSSLPTAQPLTSREASVVDGSAVTLVAQALESDSVRGKGLEIDSVHEKGLEVNS APDHTGNSWTIQLEAAPSKGEAELSLKNEARSLGPLCYQIPNMEDVVPVSSSLFSDHL TGNHTSEHCGDDILSPAGCTTPPPTKGKLTSQLSVDSILKSAANSFPGTPSILKRRKR DKSTPVSASEMKISGSNTDRFYTPMGMEPATATPESFKTTSFLSLGSLDGSVKSFDVS PQYRARSKRMALTKTVEKQLDFSSDGLDTCGSEILNSSCNNSQSTLSITEAPKLKEKE HAVQLENLTKNFAHTTNLDVT</aaseq> |
| Gene Sequence |
<dnaseqindica>458..627#1738..1789#2144..2201#2607..2701#2787..2890#3443..3571#3855..5010#acgggcgaggttttttgaaagcatcatcctctctctctcctcccccatcgactacccccgacacccaacggaagccgtagaaggagagagaggggaaaagcttgctattttataccctcatctagggttccgagcccaccggctctctccccccttgctggcttcgcgccatctctcccgccaccgcctccccccctccccccctccgcctccgatagattcgcgctcgccctcctgacccacggattcggttcccacgaaaccgctcgatctgggggatctgctagattcgtaaggcggcgtttcgatcgatctgttcgggggtttctgccgtggggaattttgatctataaagttagttttttgattttgtttcttttttgctttttggtggcggtttcgctgtgttaggtttgtggtggtggtggtggtggtgaggatgtgcttgtgccgttagatgggggccatggcgatggtggagcaggaggggtgcgtcgagaaccggcagcccctggcggcgtcgagctcgtcagtttccgacggaagcagctatggcggcggcggcggcgggctcgcgcagatgtcgccgccggtgtctagctcggcgaattccatctccggtcttaggtaaggaagttctttgcccgattcccctcagttgggatcctattatgggagggatctaggaaggtccgttggttagtacctaagttcgtgtggtagttgagaaaaatggggccttcgtggatggctccccgatggttgtgtggttttatcgctggcaatttgccctgtctgtgattccacgagcggttgtttgtgcgtttgagaaaaaatcttgccacgaggagtgctaccttggcggatatgtggcaccgagagtgttgtggtggctgtttgagtgataaatttggtgtttttttggtatccatttgatgagatacttttttatattcagacatgactttgagtgtgttatgatggatatactgatatgaaggcgttttccttttgggatggctgttgatgttttcatttctgtaattaatttgcttgccttactttaaagtgtgtatattgatgtaatttggataaaaatatgttcacttggaacactaaattggtccggttacatgcagcagtagttgtttaaactggaaatgtgttttttatgccatgacccaaggttttattagatttaatatgcttgggctacagcattggagataccatttacttgtaaccttgatgtgtatattttttgttctgactgctgcgctaaaatatggtactggtgcactatagaaatagttgttttaatgaataaacagtatttttttgttaggccctgtttggaacgtttaccctttgtgtaagattcctgagttcaaaacagggcctgaatagttgcagttacaactaaccgccatttttgtttcccacttttaatttcttgatgatgaattgaatggtgttagtagggttaagcagtatttttttcatggaaatctttcttgttcatgaaaggctccacacatgggaaatttacattgtcgaactatagccttaatgttcttccctatgtaggtctctcaaccaacaattgctgtttttttgggtcatcttatgctagtgtatttagatataccttgtgccttctcggacctaatatgctattgtgccctgaacagaaaatctttcactttatttttgctgataatctttatcttatgttaggcggacaagcgggccaataaggagagcaaagggtggctggacaccagaggaggtcaacacctcactctctattgtacttcataaatactccataagtcctattaaatgcaatacatacctacatttgttacacatatatgctagtttggtttgtatatgtattttggcatattcagtggtaatatcatgcactggaaaagttcttagttaatctttttccattcctgtttttctaaggttcttattttatccgattttatttcatatatgtttttattgttattcatggtctgaccattgattttttgtttgaatcaaatcttctattttcctgcttgaacgacatttccatgttgattcagttacacaatcactgttactaattggtcttaacttatcgcaacaggacgagacattgaggaaggcagttgaggcttataagggtagaaactggaagaaaataggtccggctctcaattccactgcaccagtgtataacttgggcagtcagttatccttgagatgatgctatcattttgttgaaaacaacctgtcaggaggattgcaaatgcctaatggtttgattccattaatatcggcccatttgcatataaatatgcgtatacaaatatgatctcagcaaactaatacatttttgcgtagattatatttgtataaatatatataagtatgcaacctatagtaatattggtgatcttgtagttgaaatagctgtttttttacctttttaatgttgtagtacttcattatatcagttgctccacgtgtactggcatacagcatgccatattatttttccatatctgtatgccaagtacattgagctcatagaatgtatctgtatgcagccgaatgttttccatacagaacagaggtacaatgcttgcatcgatggcaaaaggttcttaatcctgaacttatcaaaggtccttggactcaagaggtttgtcatgtagctgactttgctttgatggtaagcgttgacattatatcttctaattgttatattaatgccatgttttctgcaggaagatgatcaaattattgatcttgtaaagaagtatggaccaacaaaatggtctgtcatagctaaggcattacctggccgtatagggaagcaatgtcgagagaggtaatatttttatatcatggctgtattgttaccaggattcagctcactgttattaataaaacttttaaatttccagttatttttttcacatatgcatttatttccctttacatgagacattaggaaacttttgtaattgcaagaaagaactttaaattttaggatgctcatagttgctacaaaagctcaggatcccgtctattatgaaatatgaattaattaaggccttgttcggttaatccctgtgatggagggattggaggggatttattccacacctattgtggtgtggaattattccccctcaatccccttcaatcctcttcaactctaaaccgaacaaggcctaagaaatgcactacttactgaaagaactgtgaaaataattggctaataattatgctacagtatctcatagctgaattcaaactgttgtccgcatataatttactttctttatggcttcaatagtatttgttatgaaaaaagtgcttaagtttggcatgatacaatacattatacggagctaagtatgaagtcatacaatcttaggtggcacaatcatctaaatccagaaataaggaaagatgcttggactactgaggaggaacaggcactcataaatgctcatcggatttatgggaataaatgggcagagatagcaaaagttcttcctggaaggtacgtttataaagcttacttccctcggaattttttgctttcattagttcatcgagctaggtcaaaatcacgctgccctcgatctcttagcgcgctgttgcaatttagtttgtttgatttttgtttcaagttctactttcaactgcttatatacctgtgcgatggtcttaaactatttaatattggttggttgttcgtaaatcagaattatgagttgtgttattattttgaaaagacacactttacatcaccttcacagtgcttcgtatgttgctttttccaggactgataattctataaagaatcattggaatagttctctcagaaagaagcaagatatgtacaataccagcaataatatggtagttccgaaactgcttgttcatgataagttcaaggataaaccaaagctgatggctatggagggtcatcttgatttgaacaaggcacccattatcaattcaaaagaccaacctggaacagctcatcggtctaattgctcagggtttctatctcgttcctcattaccaactgctcagccacttacttcccgtgaggcatctgtagttgatggttccgctgtcactttagtggcgcaagctctggaaagtgattctgtccgtggtaagggcttggaaattgattctgttcatgagaaaggtcttgaggttaactcggcacctgatcataccgggaactcttggaccattcaactagaagctgcaccatccaaaggtgaagcagaattatctttgaagaatgaggcacgctctcttggtccactttgttaccagatccctaacatggaagatgtagtccctgtgagttcctcactcttttctgaccatcttactgggaaccatacatctgaacattgtggggatgatatattgtcacctgctggctgcactactccccctccaacaaagggaaaattaacaagccagcttagtgttgattcaattttgaagagtgctgctaacagttttccaggtactccttcaatattaaaaagaagaaaacgggataaatcaacacctgtttcagctagtgagatgaagatcagtggatcaaatactgatagattttacactcccatggggatggaacctgctacagctacaccagagtcattcaaaactacatcttttttgtcattgggttctcttgatggttctgtaaagagctttgatgtttcaccacaataccgggcaaggtcaaaaagaatggctctcacgaaaacagttgagaaacagctggatttttcatcagatggattagatacttgtggttctgagattctgaactcttcctgcaataattcccaaagcacactttctattaccgaagccccaaaactgaaagaaaaggaacatgctgttcaattggaaaatttaaccaagaactttgcgcatacaaccaatttggatgtaacctaatcttggaaaatgcaggtatgctgttacaaaaatcttgaaaacccctgctttacaattgagatgcattatacttttcatagccagtctacaaattctacctggtttatctgatccatgttggtagttgggtccttttattaattgatttgaacatggaagaagtgtgcataattgttttaccattgccttgtaggaactgacatcaatctgatgaagtaggacagatgagctgaagtgtctttctgagctgatgagctttttctcaaagtatcacgattaaaccacaacaagatcaaacaaaaccggtggagaagaattccagaattgaagctttgtactgagtacagaaggtccaccaatcattctcccatctcttctaaccaaggtgtacaaaagatgagcgacactctgtagaagaatacattgctcgctgcactttgtgaatttacagttagaaaattcagattgtagaagccacaggtgtgtacaaaacaacaacgaaaaaaaattatagtgttagaacgtcattatagctgaataagagtattcctcattggattgtcagatttattttgtcttgtgctttccctttgtcgcgaaacagccagggtctgctctgtgtaacaataataaaacccgtttttgc</dnaseqindica> 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| External Link(s) |
