籽粒高效特异性表达启动子的克隆与表达分析

Cloning and Expression Analysis of Grain Specific Expression Promoter

启动子是调控基因表达的重要元件,本研究从燕麦中克隆了燕麦球蛋白基因的启动子序列,经过比对发现新克隆的启动子序列长960 bp,与参考序列AY795082存在9个SNPs。对启动子组成元件进行分析表明,该启动子含有胚乳特异表达所必须的5个Skn-1(GTCAT)基序;在启动子的正链第622 bp和830 bp处存在胚乳表达相关的顺式调控元件GCN4;在启动子的正链550 bp和负链869 bp处有两个O_(2)位点,曾在玉米中被验证为醇溶蛋白代谢顺式调控元件;在启动子的869 bp处有一个回文结构ACATGTCATCATGT,该序列是胚乳特异表达所必需的。除了上述与胚乳特异性表达有关的元件,该启动子序列中还有许多与逆境响应有关的序列元件。利用上述启动子驱动GUS基因表达试验证明,该启动子启动功能基因表达的强度约为参考序列的5倍,为利用转基因技术改良小麦籽粒性状提供了良好的基因表达驱动元件。

Promoter is an important element for gene expression regulation.In this study,the promoter sequence of oat globulin gene was cloned.After alignment,it was found that the length of the new cloned promoter sequence was 960 bp.There were 9 SNPs with reference sequence AY795082.Analysis of the promoter components showed that the promoter contained five Skn-1(GTCAT)motifs necessary for endosperm specific expression.There were cis regulatory elements GCN4 related to endosperm expression at 622 bp and 830 bp of the positive chain of the promoter.There were two O2 sites at 550 bp of positive chain and 869 bp of negative chain of the promoter,which had been verified as the cis regulatory element of gliadin metabolism in maize.There was a palindrome structure ACATGTCATGT at 869 bp,which was necessary for endosperm specific expression.In addition to the above elements related to endosperm specific expression,there were many other elements related to stress response in the promoter sequence.The experiment of GUS gene expression driven by the promoter showed that the intensity of GUS gene expression was about 5 times of the reference sequence,which provided a very good gene expression driving element for improving wheat grain traits by transgenic technology.