大豆ABA信号途径GmCDPK SK5基因异源表达探究

Study on the Heterologous Expression of GMCDPK SK5 Gene in Soybean ABA Signaling Pathway

为探究大豆CDPKI家族成员GmCDPK SK5参与防御逆境胁迫的途径,本研究分析外源ABA作用不同时间对大豆中GmCDPK SK5基因表达量的影响,克隆基因上游启动子序列并分析其顺式作用元件,构建GmCDPK SK5pro∷GUS融合植物表达载体,转化烟草和拟南芥,并通过GUS组织化学染色和定量表达分析方法研究GmCDPK SK5启动子对外源施加的Ca^(2+)和ABA的响应。结果显示:施加外源ABA能够诱导GmCDPK SK5基因下调表达。启动子顺式作用元件分析表明该启动子含有3个TATA盒和23个CAAT盒基本转录元件和多种与胁迫相关的顺式作用元件。烟草瞬时表达系统中,Ca^(2+)浓度增加,GUS酶活性增加,在50 mmol·L^(-1)Ca^(2+)条件下,处理组中GUS酶活性是对照的2.1倍;ABA浓度增加,GUS酶活性减弱,在0.1μmol·L^(-1)ABA条件下,对照组中的GUS酶活性是处理的5倍;ABA能诱导GmCDPK SK5基因下调表达。在拟南芥中,与WT和cpk4-1突变体株系相比,在外源施加ABA的条件下,GmCDPK SK5过表达株系在种子萌发、幼苗生长和气孔运动等方面表现得最敏感。3个株系中与ABA相关的基因ABF2、ABF4、ABI5、RD29A、RAB18和KIN1差异表达。结果说明GmCDPK SK5启动子对外源施加的Ca^(2+)和ABA有响应,且GmCDPK SK5通过影响与响应ABA相关基因的表达来增强植株对ABA的敏感性。

In order to explore the ways of CDPKI family member GmCDPK SK5 participating in the defense against adversity stress,in this study,the promoter sequence of GmCDPK SK5 gene was cloned,and the GmCDPK SK5 pro∷GUS fusion plant expression vector was constructed to transform tobacco and Arabidopsis.The results showed that the application of exogenous ABA induced down-regulation of GmCDPK SK5 gene expression.Analysis of promoter cis-acting elements showed that the promoter contains 3 TATA boxes and 23 CAAT boxes basic transcription elements and a variety of cis-acting elements related to stress.GUS histochemical staining and quantitative expression analysis were used to study the response of GmCDPK SK5 promoter to exogenous Ca^(2+) and ABA.The results showed that the activity of GUS enzyme increased with the increase of Ca^(2+) concentration.Under the condition of 50 mmol·L^(-1)Ca^(2+),the activity of GUS enzyme in the treatment group was 2.1 times of the control group.While the GUS activity decreased with the increase of ABA concentration.Under 0.01μmol·L^(-1)ABA,the GUS activity in the control group was 5 times of the treatment group.Therefore ABA can induce the down-regulation of GmCDPK SK5.In Arabidopsis,compared with WT and cpk4-1 mutant lines,GmCDPK SK5 overexpression lines were the most sensitive to exogenous ABA in seed germination,seedling growth and stomatal movement.The ABA-related genes(ABF2,ABF4,ABI5,RD29 A,RAB18 and KIN1)in the three lines were differentially expressed.The results showed that GmCDPK SK5 promoter responds to exogenous Ca^(2+) and ABA,and GmCDPK SK5 enhances the sensitivity of plants to ABA by affecting the expression of ABA-related genes.