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淀粉的合成是一个复杂的生化过程。sbeⅡa基因是淀粉合成过程中的关键酶基因之一,在小麦籽粒成熟过程中对淀粉,尤其对支链淀粉的生物合成调控起重要作用。为了解sbeⅡa基因的调控机理及其胚乳特异表达特性,利用APCR方法克隆了sbeⅡa启动子3094 bp片段,并对该片段进行测序,同时利用GUS瞬间表达体系对sbeⅡa启动子序列进行不同片段的功能缺失研究。研究结果表明,3094 bp序列(存在于sbe.g融合体中)具有稳定的启动子活性,而5＇或3＇缺失体的启动子活性明显降低。在sbeⅡa启动子中间缺失体中,一些缺失体仅有微弱活性,但sbe.e在缺失-1579—1210 bp片段情况下,却具有比sbe.g(含启动子全长序列)还要高的启动子活性,研究初步证明,一些固定序列(motifs),如-300 bp因子、G盒以及醇溶蛋白盒(Prolamin box)等作为正调控因子在决定启动子胚乳特异表达模式中是必需的,而且在-1579—1210 bp片段中也存在一些负调控因子或固定序列。在瞬间表达检测体系中,小麦胚乳组织年龄对检测结果有着重要影响。
The synthesis of starch is a complex biochemical process. sbeⅡa is one of the key enzyme genes in starch synthesis. It plays an important role in regulating the biosynthesis of starch, especially amylopectin, during grain maturation in wheat. In order to understand the regulatory mechanism of sbeⅡa gene and the specific expression characteristics of endosperm, the 3094 bp fragment of sbeⅡa promoter was cloned by APCR method and the fragment was sequenced. At the same time, the functional deletion of sbeⅡa promoter sequence was performed by GUS transient expression system the study. The results show that the 3094 bp sequence (present in the sbe.g fusion) has a stable promoter activity while the 5 ’or 3’ deletion has a significantly reduced promoter activity. In the intermediate deletion of the sbeIIa promoter, some of the deletions showed only weak activity, but sbe.e had a higher deletion than the sbe.g (containing the full-length promoter sequence) in the absence of the -1579-1210 bp fragment Promoter activity, preliminary studies have shown that some motifs, such as the -300 bp factor, G box and prolamin box, as positive regulators in the determination of promoter endosperm-specific expression mode is necessary, Moreover, there are some negative regulators or fixed sequences in -1579-1210 bp fragment. In the transient expression detection system, the age of wheat endosperm tissue has an important impact on the test results.