大豆转录因子GmNAC8的克隆及耐旱性功能分析

Cloning and function analysis of GmNAC8 transcription factor gene response to drought stress

大豆转录因子GmNAC8在耐旱材料和敏感材料中明显差异表达,该基因CDS序列全长1 092bp,编码363个氨基酸残基。通过软件预测,蛋白质相对分子质量为41.82k D,等电点为8.51,其N-端含有42aa组成的NAC保守结构域,C-端高度变异。进化树分析表明,该蛋白与菜豆、红豆、绿豆同源关系最近。在洋葱表皮细胞中的亚细胞定位分析表明GmNAC8蛋白定位于细胞核。转录水平表达分析表明,GmNAC8基因在转基因株系叶中的表达量要明显高于根中的表达量;GmNAC8基因受0.1mmol/L IAA和ABA诱导表达量显著上升,受GA和SA抑制。GmNAC8基因在拟南芥中超量表达可以使拟南芥叶片更绿,耐旱能力增强。抗旱生理指标检测表明,在干旱处理10d后,转基因拟南芥叶中蛋白质含量、脯氨酸含量、POD活性均高于野生型拟南芥,丙二醛含量明显低于野生型拟南芥,仅为42%。本研究结果为进一步研究GmNAC8在植物耐旱反应中的分子机理奠定基础。

In order to obtain functional genes in response to drought stress,a DGE（ digital gene expression）profile,two drought- tolerance and sensitivity materials under drought stress,had been constructed in our previous study. A gene encoding NAC tanscription factor,named as GmNAC8,was chosen as candidate gene from DGE profile. The gene had CDS full- length sequence（ 1 092bp）,and encodes 363 amino acid. By software analysis,its protein had a molecular mass of 41. 82 k D and PI 8. 51. The N- terminal aa residue of GmNAC8 contains a typical NAC conserved domain with 42 aa,and C- terminal aa residue is highly variable. Homology tree demonstrated that GmNAC8 shared high homology with Phaseolus vulgaris,Vigna angularis and Vigna radiata. Subcellular localization revealed that GmNAC8 was expressed in nucleus. Transcription analysis by qRT- PCR showed that the expression of GmNAC8 in leaf was apparently higher than that in root from transgenic lines. GmNAC8 was up- regulated by 0. 1mmol / L IAA and ABA,and was down- regulated by 0. 1mmol / L GA and SA. The leaves of transgenic Arabidopsis thaliana lines were greener than CK,and drought tolerance was improved under water- deficient condition by overexpression of GmNAC8. Drought- tolerant physiological analysis revealed that the contents of protein,proline,and POD activity were higher in transgenic lines in 10 days drought treatment,while malonaldehyde（ MDA）was significantly lower,only occupied 42%. This study would provide understanding of the molecular mechanisms of plants response to drought.