OsCPK12基因功能研究和互作蛋白筛选

Gene function of OsCPK12 and screening of its interaction proteins

通过反向遗传学研究方法,构建OsCPK12基因敲除和超表达突变体,用150 mmol/L的NaCl进行盐胁迫处理,结果显示:超表达株系无论株高、根长还是叶片状态都优于野生型,而基因敲除突变体相比于野生型株高明显矮化,根的发育形态差,叶片枯黄,证实OsCPK12对水稻耐盐有正调控作用。用real-time PCR对突变体株系的根、叶鞘、叶片多个部位的激素信号转导路径基因的表达量进行分析,结果显示:超表达株系的多个激素受体编码基因和下游调控基因表达量显著上调,说明OsCPK12参与激素的信号转导并影响水稻抗逆反应。为进一步研究OsCPK12的基因功能,通过在水稻原生质体中表达融合蛋白进行OsCPK12的亚细胞定位,确定OsCPK12定位于质膜。用酵母双杂交的方法在水稻中花11膜蛋白文库中筛选到2个水通道蛋白OsPIP1-1和OsPIP2-7,且酵母点对点验证为阳性,推测OsCPK12可能通过与OsPIP1-1和OsPIP2-7互作,调控水分子进出细胞,提高了水稻的耐盐性。

The knockout and overexpression mutants of OsCPK12 were constructed with reverse genetics.The plant height,root length and leaf phenotype of the over-expressed lines was much better than that of the wild type under the 150 mmol/L NaCl treatment.The plant height of knockout mutants was shorter,the root development were affected,and the leaves were withered and yellow compared with the wild type,indicating that OsCPK12 had positive regulation effect on rice salt tolerance.The results of the expression level of hormone signal transduction pathway genes in different tissues of mutant lines analyzed by real-time PCR showed that expression of multiple hormone receptor coding genes and downstream regulatory genes in the overexpression lines were significantly upregulated compared with the wild-type.It is indicated that OsCPK12 was involved in signal transduction of hormones,which affected rice stress response.Subcellular localization of OsCPK12 was done by fusion expression of target protein with fluorescent protein to further characterize the gene function of OsCPK12 and explain the mechanism.OsCPK12 was proved to be mostly located in the plasma membrane,laying the foundation for further research.Two water channel proteins,OsPIP1-1 and OsPIP2-7 were screened from Zhonghua 11’s membrane protein library and its interaction with OsCPK12 was further confirmed with yeast two-hybrid test.Results indicated that OsCPK12 may interact with OsPIP1-1 and OsPIP2-7 and regulate the entry and exit of water molecules which improve the rice salt tolerance.