玉米耐旱相关基因ZmMKKK18的功能鉴定及响应干旱胁迫的作用机理研究

In this study, eight MAPKKK genes have been identified to show a significant differences of the expression level responsive to drought stress in maize using the in silico analysis and RNA-Seq technology. Further qRT-PCR analysis in 11 maize inbred lines indicated, among 8 genes, ZmMKKK18 showed the significantly up-regulated trend in response to drought stress. Correlation analysis revealed that there was a significant correlation between expression levels of ZmMKKK18 and relative biomass responsive to drought. Association analysis between genetic variation of ZmMKKK18 gene and plants drought tolerance at seedling stage in a natural variation maize population showed that the genetic variation of ZmMKKK18 was significantly associated with drought tolerance of plants. In this study, we will analyze the biological function with respect to drought tolerance of target gene through the expression pattern analysis of ZmMKKK18 responsive to drought stress, the over-expression of ZmMKKK18 and gene knock-out analysis in maize. The correlation of ZmMKKK18 gene sequence variation with plant drought tolerance will be analyzed in various maize inbred lines to identify the genetic effect of ZmMKKK18 on drought tolerance. Meanwhile, the transcription regulation of ZmMKKK18 and interaction protein analysis responsive to drought stress will be studied to investigate the mechanism of drought stress tolerance of ZmMKKK18 gene.

利用生物信息学技术、RNA-Seq转录组测序方法获得了8 个干旱胁迫显著差异表达的玉米MAPKKK 基因。对11个玉米自交系进一步RT-PCR分析表明，ZmMKKK18基因表达水平差异极显著，且与植株生物量的耐旱系数呈显著相关。利用玉米自然变异群体对ZmMKKK18基因的遗传变异和苗期耐旱性表型的关联分析发现，该基因的遗传变异和苗期耐旱性显著关联。本研究拟通过分析ZmMKKK18基因的玉米干旱响应的精细表达模式、开展该基因在玉米中的过表达及基因敲除研究，以及通过关联分析与连锁分析方法分析ZmMKKK18基因与植株耐旱性的关系，鉴定ZmMKKK18基因在干旱胁迫逆境下的的生物学功能；同时通过转录调控分析以及干旱应答调控的互作蛋白鉴定分析等方法，明晰玉米ZmMKKK18基因响应干旱胁迫的作用机理。

干旱是玉米生产中面临的一个重要逆境胁迫因子，通过鉴定优良抗旱基因资源，并利用这些基因资源对玉米的抗旱节水性状进行遗传改良，可为玉米产业的可持续发展提供重要支撑。本研究以潜在的玉米耐旱基因ZmMKKK18为研究对象，通过荧光PCR分析发现该基因在玉米幼苗期、开花吐丝期叶片、根、雄穗、雌穗组织均有表达，叶片表达量最高，根、雄穗表达量较低，耐旱性品种叶片中该基因对干旱响应上调幅度更加显著。亚细胞定位结果表明ZmMKKK18基因的表达部位位于植物细胞的叶绿体内。通过过表达及基因敲除实验发现，ZmMKKK18基因过表达的植株耐旱性强于野生型植株，进一步鉴定试验发现干旱胁迫条件下过表达植株可以维持更高的光合效率和水分利用效率，而敲除ZmMKKK18基因则使得植株对干旱胁迫更为敏感。转录组分析发现，在干旱胁迫条件下ZmMKKK18基因主要通过调控NAC转录因子上调以减缓干旱刺激的HSP及PP2C相关基因的大量表达，从而使过表达植株能够有效抵御干旱刺激造成的内质网胁迫，增强了植株的耐旱性。通过酵母双杂交获得1个与ZmMAPKKK18 存在互作与细胞壁稳态维持等途径相关的MAPK 家族新基因GRMZM2G162333，推测可能在ZmMAPKKK18基因参与的MAPK 级联反应传递干旱胁迫应答信号中起作用。本研究对于玉米的抗旱性改良，增强玉米对干旱等逆境的抵御能力，具有非常重要的意义。