参与ABA诱导的水稻OsCaMs基因鉴定及其功能分析
发布时间：2018-11-16 15:54
【摘要】：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。
[Abstract]:The transient expression vector of OsCaMs was constructed by using Xudao 4 as the material. Five OsCaMs quantitative primers were designed by bioinformatics and RT-PCR methods. The OsCaMs gene involved in ABA induction was identified and its physiological function was analyzed. To lay a foundation for the further study of the core components of ABA signal transduction. The results showed that: (1) the CDS of the five family genes of rice OsCaMs was 450bp. ABA (100 渭 mol L ~ (-1) could significantly induce the expression of OsCaM1-1 and OsCaM1-2. (2) OsCaM1-1-YFP and OsCaM1-2-YFP expressing fluorescent protein were transformed into protoplasts by PEG. Laser confocal analysis showed that the two genes were located in the nucleus, cytoplasm and cell membrane. The interference vectors of OsCaM1-1 and OsCaM1-2, dsOsCaM1-1 and dsOsCaM1-2. (3), were constructed. The antioxidant protective enzymes APX and SOD induced by ABA in rice protoplasts were significantly increased by 35% and 31%, respectively. The transient expression of protoplasts and transient silencing system showed that both OsCaM1-1 and OsCaM1-2 could affect the activities of antioxidant protective enzymes APX and SOD. The transient overexpression of these two genes in protoplasts increased the activity of APX and SOD by 1.15 ~ 1.45 times. (4) exogenous H_2O_2 (10mmol L-1) pretreatment could induce the up-regulation of OsCaM1-1 and OsCaM1-2 gene expression in rice seedlings. OsCaM1-2 could induce the accumulation of H_2O_2 in the protoplasts of rice. (5) the transient expression of OsCaM1-1 and OsCaM1-2 in the protoplasts of rice could induce the expression of OsrbohB and OsrbohE, respectively. Moreover, transient expression of OsrbohB and OsrbohE in rice protoplasts could induce the expression of OsCaM1-1 and OsCaM1-2, respectively, indicating that there was a positive feedback regulation mechanism between OsCaMs and Osrbohs. The results showed that OsCaM1-1 and OsCaM1-2 were the homologous genes involved in ABA signal transduction. OsCaM1-1 and OsCaM1-2 were induced not only by ABA but also by H_2O_2, and ABA was regulated by H_2O_2.
【作者单位】： 南京农业大学生命科学学院;
【基金】：基金项目:中央高校基本业务费(KYZ201157)
【分类号】：Q943.2
，
本文编号：2335943
[Abstract]:The transient expression vector of OsCaMs was constructed by using Xudao 4 as the material. Five OsCaMs quantitative primers were designed by bioinformatics and RT-PCR methods. The OsCaMs gene involved in ABA induction was identified and its physiological function was analyzed. To lay a foundation for the further study of the core components of ABA signal transduction. The results showed that: (1) the CDS of the five family genes of rice OsCaMs was 450bp. ABA (100 渭 mol L ~ (-1) could significantly induce the expression of OsCaM1-1 and OsCaM1-2. (2) OsCaM1-1-YFP and OsCaM1-2-YFP expressing fluorescent protein were transformed into protoplasts by PEG. Laser confocal analysis showed that the two genes were located in the nucleus, cytoplasm and cell membrane. The interference vectors of OsCaM1-1 and OsCaM1-2, dsOsCaM1-1 and dsOsCaM1-2. (3), were constructed. The antioxidant protective enzymes APX and SOD induced by ABA in rice protoplasts were significantly increased by 35% and 31%, respectively. The transient expression of protoplasts and transient silencing system showed that both OsCaM1-1 and OsCaM1-2 could affect the activities of antioxidant protective enzymes APX and SOD. The transient overexpression of these two genes in protoplasts increased the activity of APX and SOD by 1.15 ~ 1.45 times. (4) exogenous H_2O_2 (10mmol L-1) pretreatment could induce the up-regulation of OsCaM1-1 and OsCaM1-2 gene expression in rice seedlings. OsCaM1-2 could induce the accumulation of H_2O_2 in the protoplasts of rice. (5) the transient expression of OsCaM1-1 and OsCaM1-2 in the protoplasts of rice could induce the expression of OsrbohB and OsrbohE, respectively. Moreover, transient expression of OsrbohB and OsrbohE in rice protoplasts could induce the expression of OsCaM1-1 and OsCaM1-2, respectively, indicating that there was a positive feedback regulation mechanism between OsCaMs and Osrbohs. The results showed that OsCaM1-1 and OsCaM1-2 were the homologous genes involved in ABA signal transduction. OsCaM1-1 and OsCaM1-2 were induced not only by ABA but also by H_2O_2, and ABA was regulated by H_2O_2.
【作者单位】： 南京农业大学生命科学学院;
【基金】：基金项目:中央高校基本业务费(KYZ201157)
【分类号】：Q943.2
，
本文编号：2335943
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2335943.html
最近更新
教材专著
