糜子抗旱节水相关基因PmMYB的克隆及表达分析

Cloning and expression analysis of drought-tolerant and wa-ter-saving related gene PmMYB in broomcorn millet

根据在糜子抗旱节水分子基础研究中获得的一个糜子MYB基因的EST序列,以其序列及水稻MYB18基因的序列为基础设计引物,扩增得到1739bp的全长基因组序列。序列分析表明,其包含121bp（347～467bp）和93bp（599～691bp）的两个内含子,3个外显子;全长cDNA序列为1525bp,其中3′非翻译区为212bp,5′非翻译区为41bp,编码区为1272bp,共编码424个氨基酸,C-端存在一个丝氨酸（Ser,S）丰富区。该基因具有两个典型的MYB类转录因子基因的DNA结合区（DNA-binding domain）,分别为13～63、66～114位氨基酸,属于典型的R2R3-MYB转录因子。对其与水稻、玉米、火炬松、拟南芥、辣椒、陆地棉、大麦及茄子等9种植物的MYB基因的R2、R3重复区的氨基酸序列多重比较,表明R2R3重复序列在植物中具有较高的保守性;基于氨基酸序列的编码区系统进化树分析表明,不同植物的MYB基因遗传分化很大,序列相似性为32%～84%,其中糜子MYB基因与水稻的MYB18相似程度最高（84%）,与大麦和玉米的相似性分别为46%和41%。通过半定量RT-PCR对其表达模式分析表明,该基因在水分胁迫和干旱后复水条件下上调表达,与糜子抗旱节水紧密相关。该基因的克隆为进一步探讨利用该基因改良其他植物的抗旱节水性奠定了良好的基础。

Exploring novel genes and gene combinations are of primary importance in plant drought-tolerance and water-saving research. Based on the EST sequence of MYB gene obtained in the research on molecular basis of drought-tolerance and water-saving in broomcorn millet （Panicum miliaceum L.）, a 1 739 bp genomic sequence of MYB gene, designated PmMYB, was amplified using primers based on this EST sequence and that office MYB18. Sequence analysis indicated that PmMYB consisted of 2 introns, 121 bp （347-467 bp） and 93 bp （599-691 bp） in length respectively, and 3 extrons. The full-length cDNA sequence of PmMYB is 1 525 bp, which includes 212 bp 3′ untranslated region, 41 bp 5′ untranslated region, and 1 272 bp coding region which encodes 424 amino acids with a serine-rich region. PmMYB is a typical R2, R3-MYB transcription factor with its 2 typical DNA-binding domains （amino acid sites of 13-63 and 66-114）.The multiple alignment of R2, R3 amino acid repeat regions of MYB genes among broomcom millet, rice, corn, loblolly pine, Arabidopsis, capsicum, upland cotton, barley and aubergine indicates that R2, R3 repeat regions are very conserved. The phylogenetic analysis based on amino acid sequence shows that MYB genes are highly divergent with similarity ranging from 32% to 84%. PmMYB has the highest similarity （84%） with MYB18 gene of rice, whereas 46% and 41% with barley and corn, respectively. Semi-quantitative RT-PCR confLrmed that PmMYB was upregulated by water stress and re-watering in broomcom millet. Cloning this gene may provide a good foundation to improve the drought-tolerance and water-saving in broomcom millet as well as in other plants