ThMYB3和ThDof2对ThVHAc1基因表达的调控

Upstream regulators of ThVHAc1

ThVHAc1基因能响应干旱、盐、重金属等胁迫诱导,该基因能有效提高转ThVHAc1基因酵母的多种抗逆能力。为进一步研究ThVHAc1基因的抗逆调控机理,本研究利用酵母单杂交技术钓取ThVHAc1可能的上游调控因子,并利用基因枪瞬时共转化技术进行初步验证。酵母单杂交结果显示,ThMYB3基因能识别ThVHAc1基因上游MYB顺式作用元件和含有MYB元件的启动子片段。ThDof2基因能识别ThVHAc1基因上游DOF顺式作用元件和含有DOF元件的启动子片段。但ThMYB3和ThDof2均不能识别突变后的元件及含对应突变元件的启动子片段。将ThMYB3和ThDof2分别构建成效应载体,各元件、突变元件、启动子片段及突变的启动子片段分别构建报告载体进行共表达验证。结果发现:效应载体ThMYB3和含MYB元件及含MYB元件的ThVHAc1启动子片段的报告载体,ThDof2和含DOF元件及含DOF元件的ThVHAc1启动子片段的报告载体瞬时共表达烟草叶片能观察到GUS染色,且GUS活性较高;而与相应突变元件及片段的共转化则观察不到烟草叶片的GUS染色,且GUS活性很低。说明只有非突变的元件和启动子片段才能与效应载体进行互作识别,激活GUS基因的表达,验证了酵母单杂交获得的上游调控基因的识别特异性。表明ThMYB3和ThDof2可能通过与相应启动子元件的结合调控ThVHAc1基因。

ThVHAc1 gene was reported to be responsive to drought,salt and heavy metal stresses,and overexpressed ThVHAc1 yeast could improve effectively multi-stress tolerance. To further understand the stress regulation mechanism of ThVHAc1,we screened the upstream regulators that could specifically recognize the cis-elements DOF and MYB in the ThVHAc1 promoter by yeast one-hybrid assay. The results showed that the ThVHAc3 gene could specifically recognize the MYB motif and the promoter segment that contains MYB motif,and the ThDof2 gene could specifically recognize the DOF motif and the promoter segment that includes DOF motif in the ThVHAc1 promoter. However,ThVHAc3 and ThDof2 could not recognize the mutated motifs and promoter segments containing these mutated motifs. Then ThVHAc3 and ThDof2 were independently constructed as effecter vector,and MYB and DOF motifs as well as their mutates and promoters were independently constructed as reporter vectors for co-expression assays. And the results of co-transient expression of the effecters and reporters confirmed that ThDof2 and ThVHAc3 can specifically bind to the ThVHAc1 promoter. ThVHAc3 and reporters containing MYB motifand ThVHAc1 promoter segments including MYB motif,ThDof2 and reporters containing DOF motif and the ThVHAc1 promoter segments including DOF motif could stain the GUS expression in tobacco leaves and showed high GUS activities. However,other co-expression referred to mutated motifs could not activate the GUS expression,indicating that only normal cis-elements and promoter segments could be recognized by effecters to activate the GUS expression. Our study suggests the effectiveness of ThVHAc3 and ThDof2 as ThVHAc1 upstream regulators.