参与ABA诱导的水稻OsCaMs基因鉴定及其功能分析

Functional Analysis and Gene Identification of the OsCaMs Involved in ABA-inducing

以‘徐稻4号’为材料,构建OsCaMs的瞬时表达载体,利用生物信息学和RT-PCR方法设计OsCaMs定量引物5个,鉴定参与ABA诱导的OsCaMs基因并分析其生理功能,为进一步揭示ABA信号转导核心组分的研究奠定基础。结果显示:(1)水稻OsCaMs的5个家族基因的CDS等长,均为450bp,ABA(100μmol·L^(-1))处理能够明显诱导OsCaM1-1和OsCaM1-2的表达。(2)利用瞬时表达载体将表达荧光蛋白的OsCaM1-1-YFP和OsCaM1-2-YFP通过PEG方法转化到原生质体中,激光共聚焦分析显示,这2个基因均定位于细胞核、细胞质和细胞膜中;并构建了OsCaM1-1和OsCaM1-2的干扰载体dsOsCaM1-1和dsOsCaM1-2。(3)经ABA诱导的水稻原生质体抗氧化保护酶APX和SOD活性分别显著提高35%和31%;原生质体瞬时表达和瞬时沉默体系分析表明,OsCaM1-1和OsCaM1-2均能够影响抗氧化保护酶APX和SOD的活性,其中原生质体中瞬时过表达这2个基因对APX和SOD活性上调达到1.15~1.45倍,瞬时干扰这2个基因对APX和SOD活性下调达到25%~30%。(4)外源H_2O_2(10mmol·L^(-1))预处理水稻幼苗可诱导OsCaM1-1和OsCaM1-2基因表达量上调,并且OsCaM1-2能够诱导水稻原生质体中H_2O_2的积累。(5)原生质体瞬时表达分析发现,在水稻原生质体中瞬时表达OsCaM1-1和OsCaM1-2可分别诱导OsrbohB和OsrbohE的表达;而且在水稻原生质体中瞬时表达OsrbohB和OsrbohE可分别诱导OsCaM1-1和OsCaM1-2的表达,表明OsCaMs与Osrbohs之间存在正反馈调节机制。研究表明,OsCaM1-1和OsCaM1-2为水稻参与ABA信号转导中具有调控抗氧化保护作用的同源基因;OsCaM1-1和OsCaM1-2不仅受ABA诱导,也受H_2O_2诱导,而且ABA是通过H_2O_2进行调控。

We used Xu rice No.4 as materials,Oryza sativa's transient expression vectors were constructed and the method of bioinformatics and RT-PCR were used to design the semi-quantitative and quantitative primers of five OsCaMs.We identified the OsCaMs gene induced by ABA and analyzed its physiological function to lay the foundation for further research on the core components of ABA signal transduction.(1)The results showed that their CDS are 450 bp.Treatment with 100μmol·L^(-1) ABA could induce the gene expression of OsCaM1-1 and OsCaM1-2 obviously.(2)The vectors(35S-OsCaM1-1-YFP,35S-OsCaM1-2-YFP)were then transformed into rice protoplasts by PEG method.Subcellular localization analysis by con-focallaser microscopy showed that the two genes are located in the nucleus,cytoplasm and plasma.Then the double-str and ed RNA interference(RNAi)of OsCaM1-1 and OsCaM1-2 were constructed.(3)The analysis of transient expression and RNAi silencing of OsCaM1-1 and OsCaM1-2 in rice protoplasts indicated that OsCaM1-1 and OsCaM1-2 required for ABA-induced have an obvious influence on the activities of ascorbate peroxidase(APX) and superoxide dismutase(SOD).The activities of antioxidant protective enzymes APX and SOD in rice protoplasts were significantly increased by 35% and 31%,respectively,after ABA induction,overexpressing the two genes in the protoplasts resulted in a fold increase in APX and SOD activities between 1.15 and 1.45 fold.APX and SOD activities were reduced by between 25% and 30% when silencing OsCaM1-1 and OsCaM1-2.(4)Treating the rice with H_2O_2,we found that exogenous H_2O_2(10mmol·L^(-1))treatment can induce gene expression of OsCaM1-1 and OsCaM1-2.And overexpressing OsCaM1-2in protoplast increased the production of H_2O_2.(5)The analysis of transient expression in rice protoplasts showed that overexpression of OsCaM1-1 and OsCaM1-2 can induce gene expression of OsrbohB and OsrbohE;while overexpression of OsrbohB and OsrbohEcan induce gene expression of OsCaM1-1 and OsCaM1-2.So between OsCaMs and Osrbohs exists a positive feedback regulation mechanism.Overall,OsCaM1-1 and OsCaM1-2 are homologous genes involved in the regulation of antioxidant defense by ABA,these 2 genes can regulate the activities of oxidative protective enzymes in ABA signaling.OsCaM1-1 and OsCaM1-2 are induced not only by ABA,but also by H_2O_2 and they are induced by ABA through H_2O_2.