Pinb基因启动子生物信息学分析及载体构建

Bioinformatics analysis of Pinb gene promoter and construction of report gene expression vector

运用生物信息学预测并分析小麦硬度基因Pinb的启动子,利用无缝克隆构建胚乳特异表达载体.采用生物信息学软件预测Pinb基因5’端上游调控区3056bp序列进行启动子预测与分析,克隆启动子,利用无缝克隆技术构建Pinb基因的胚乳特异表达的重组质粒,重组质粒经测序鉴定构建正确.Pinb基因5’上游-3060bp至-4bp区域内存在启动子活性,在-2419bp至-500bp间启动子活性分值均在0.8以上,其中-550bp至-500bp区启动子片段分值最高,达到0.99.在-2862bp处和-362bp处各有一个转录起始位点.在-544bp处有一个CAAT盒信号,在-541bp处有一个TATA盒信号.在-2362bp至-2256bp、-1704bp至-1559bp和-537bp至-361bp处各有一个CpG岛.取最有可能的757bp(-803bp至-47bp)启动子片段构建重组质粒(LBLV232),质粒经酶切和测序验证正确.最终,成功构建Pinb基因启动子报告基因表达载体,为系统研究该基因启动子活性提供前期基础.

Using bioinformatics to predict and analyse the promoter of Pinb gene,then constructing specific endosperm expression vector by using the Seamless Cloning technology.The bioinformatics was used to predict and analyze the promoter region of Pinb during gene 5＇upstream 3056 bp regulatory region,and then the promoters were cloned.The Pinb endosperm expression plasmids were constructed,and these recombinant plasmids were identified by DNA sequencing.The bioinformatics results show that there are promoter activity from-3060 bp to-4 bp.The activity value of-2419 bp to-500 bp is over0.8 and-550 bp to-500 bp is 0.99.There are two transcription start site boxes in-2862 bp to-362 bp,respectively.A CAAT box in-544 bp,and a TATA box in-541 bp.Three CpG islands were detected in-2362 bp to-2256 bp,-1704 bp to-1559 bp,and-537 bp to-361 bp.The most possible fragments 757 bp（-803 bp to-47 bp）was chosen to construct plasmids LBLV232.These plasmids were identified by enzyme digestion and sequencing,the size of the fragment and the sequence was correct.Based on the results of bioinformatics analysis,the successful construction of Pinbgene promoter report gene expression vector,provides a preliminary basis for detecting the gene promoter activity.