碱胁迫应答基因GsSnRK1.1与上游调控因子GsGRIK1互作功能分析

研究针对野生大豆蛋白激酶GsGRIK1与GsSnRK1.1相互作用是否参与植物响应碱胁迫,采用生物信息学、分子生物学以及基因工程等技术手段开展系统研究。首先作碱胁迫应答基因GsSnRK1.1和GsGRIK1全长基因克隆功能验证,证明其有显著耐碱功能;构建酵母表达载体PYX212和PYX242,利用酵母四重突变株(sak1/tos3/elm1/snf1)作表型分析。结果表明,共转化GsSnRK1.1和GsGRIK1的转化子可在非葡萄糖碳源和碱胁迫处理的选择培养基上正常生长。由此证明野生大豆GsSnRK1.1受上游调控因子GsGRIK1激活调控,且发现其可能参与植物耐碱胁迫应答机制。通过Western blot体外生化分析,证明上游调控因子GsGRIK1通过钙离子依赖途径磷酸化激活GsSnRK1.1。利用组织染色法,对GsSnRK1.1和GsGRIK1作碱胁迫下表达模式分析,结果表明,GsSnRK1.1和GsGRIK1基因表达显著响应碱胁迫。通过碱胁迫处理过量表达GsSnRK1.1和GsGRIK1大豆毛状根,作功能表型鉴定,结果表明,共表达GsSnRK1.1和GsGRIK1的大豆毛状根耐碱能力最显著。研究首次阐明GsGRIK1通过钙离子依赖途径磷酸化调控GsSnRK1.1参与碱胁迫应答通路,为完善碱胁迫应答通路奠定重要基础。

Analysis of interaction function of upstream regulator GsGRIK1 and GsSnRK1.1 in response to alkaline stress

The study was systematicly performed using bioinformatics, molecular biology and genetic engineering techniques to investigate whether the interaction between GsSnRK1.1 and GsGRIK1 was involved in the process of plants response to alkali stress. Firstly, the full-length CDS of GsSnRK1.1 and GsGRIK1 was cloned and the function was analyzed, the results showed that these two members played positive roles in alkaline stress tolerance. The yeast expression vectors PYX212 and PYX242 were constructed for phenotypic analysis with yeast quadruple mutant (sak1/ tos3/ elm1/ snf1). The results showed that the yeast co-transformed with GsSnRK1.1 and GsGRIK1 in yeast cells could grow normally on non-glucose carbon sources and alkaline stress selective media. This results also demonstrated that GsSnRK1.1 was regulated by the upstream regulatory factor GsGRIK1, and it was found that the two genes might be involved in the plants alkali response pathway. It revealed that GsGRIK1 could phosphorylate GsSnRK1.1 in the calcium-dependent pathway by Western blot analysis. By GUS staining assays, and the expression patterns of GsSnRK1.1 and GsGRIK1 were analyzed under alkaline stress, the results showed that both GsSnRK1.1 and GsGRIK1 genes could significantly response to alkaline stress. The functional analyses showed that over-expression of GsSnRK1.1 or GsGRIK1 in soybean hairy roots could significantly increase the plants alkaline stress tolerance. It firstly elucidated that GsGRIK1 regulated GsSnRK1.1 through calcium dependent pathway and participated in the pathway of alkaline stress response, and the results also provided a theoretical basis for the further exposition of the alkaline stress response pathway.