Pib基因启动子内YTCANTYY暗诱导分子元件功能的转基因验证

Function Verifying of Darkness Inducing Motif YTCANTYY in Pib Promoter via Rice Transformation

用不同长度5′端缺失的Pib启动子驱动gus基因的水稻转基因植株,系统研究了Pib启动子中分子元件YTCANTYY拷贝数目与该启动子启动活性和暗诱导性的关系。GUS组织化学分析结果表明,含6个、3个和1个拷贝该分子元件的Pib启动子的转基因水稻愈伤组织在暗处理后均能在X-Gluc溶液中显示不同深浅的GUS蓝色,而6个分子元件全部缺失的启动子片段的转基因愈伤组织不显现GUS蓝色。荧光定量分析结果表明,Pib启动子序列具有很强的器官特异性,即便是长仅222bp、不含YTCANTYY元件的5′端缺失体Pib启动子-gus构建的转基因植株,其根部Pib启动子活性仍高于地上部器官。但其启动活性和暗诱导性都随启动子缺失片段的缩短即该分子元件拷贝数增加而提高。这些结果表明,YTCANTYY在Pib启动子序列中是一个暗诱导的功能性分子元件,它赋予了启动子的暗诱导性,至少在6个拷贝以内,Pib启动子的暗诱导活性与其拷贝数目呈正相关,各个YTCANTYY分子元件的暗诱导活性可能是累加的。

By using transgenic rice plants of 5＇ deletion Pib promoter fragment-gus constructions, the relationships between the copy number of motif YTCANTYY in Pib promoter and activity of Pib promoter and its darkness induction attribute were systematically analyzed. The results from GUS histochemistry analysis revealed, after darkness induction the transgenic rice calli of Pib promoter fragments harboring 6, 3, or 1 copy of YTCANTYY motif were developed GUS blue to a different degree in X-gluc solution, but the 5＇ deletion Pib promoter fragment without any YTCANTYY was unable to drive gus gene to show any GUS blue Fluorescence quantitative analysis results showed that Pib promoter sequence had strong organ specificity. Even for the 222 bp fragment of 5＇ deletion Pib promoter, which does not contain YTCANTYY motif, in its transgenic rice plants the Pib promoter activity of root was higher than that of above ground organs. But the activities of promotion and darkness inducing were increased with the increase of promoter fragments in length and the increase of this motif copy. These results indicated that YTCANTYY in Pib promoter is a functional motif for darkness induction and in other words this motif confers the darkness induction on Pib promoter. At least within 6 copies, the activity of darkness induction was positively correlated with the copy number of YTCANTYY. And the activity of darkness induction for each individual YTCANTYY motif seemed to be additive.