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为研究6-磷酸山梨醇脱氢酶(Sorbitol-6-Phosphate dehydrogenase,S6PDH)基因启动子(S6PDHp)的逆境诱导表达特性,利用Gateway技术构建了S6PDH基因启动子区5’端系列缺失体与GUS基因的融合表达载体,并通过农杆菌介导法转化拟南芥。对转基因拟南芥进行低温和外源ABA处理,通过GUS蛋白活性变化分析S6PDHp的逆境诱导表达特性。研究结果发现,通过Gateway技术构建了4个S6PDHp 5’端系列缺失体与β-葡糖苷酸酶(GUS)基因的融合表达载体(p GWB433-S6PDHp1、p GWB433-S6PDHp2、p GWB433-S6PDHp3和p GWB433-S6PDHp4)并获得了相应的转基因拟南芥。对转基因植株进行低温处理后发现,p GWB433-S6PDHp3转基因植株中的GUS活性增幅最大,达到显著水平,而其他转基因植株中的GUS活性基本保持不变。外源ABA处理后发现,除p GWB433-S6PDHp4外,其余启动子缺失体转基因拟南芥中GUS活性显著升高。以上结果表明,低温和外源ABA能够诱导S6PDHp的表达,但不同的缺失体响应程度不同,意味着在S6PDHp序列(-2396bp至-236bp)内中可能存在着响应逆境胁迫的正负调控顺式作用元件。
In order to study the stress-induced expression of the S6PDH gene promoter (S6PDHp), the 5 ’end deletion sequence of S6PDH gene promoter region was constructed with Gateway technology to detect GUS The fusion expression vector was constructed and transformed into Arabidopsis by Agrobacterium tumefaciens. The transgenic Arabidopsis thaliana was treated with low temperature and exogenous ABA, and the expression of S6PDHp induced by stress was analyzed by the change of GUS activity. The results showed that four fusion proteins of S6PDHp 5 ’end deletion and β-glucuronidase (GUS) gene were constructed by Gateway technology (p GWB433-S6PDHp1, p GWB433-S6PDHp2, p GWB433-S6PDHp3 and p GWB433-S6PDHp4) and obtained the corresponding transgenic Arabidopsis thaliana. After low temperature treatment, the GUS activity in p GWB433-S6PDHp3 transgenic plants showed the largest increase at a significant level, while the GUS activity in other transgenic plants remained basically unchanged. Exogenous ABA treatment found that, in addition to p GWB433-S6PDHp4, the rest of the promoter-deficient transgenic Arabidopsis transgenic GUS activity was significantly increased. The above results showed that low temperature and exogenous ABA can induce the expression of S6PDHp, but different degrees of response to different deletions means that positive and negative cis-acting cis-acting response to stress may exist in S6PDHp sequence (-2396bp to -236bp) Role of components.