两种HMW-GS基因启动子片段的克隆及分析

Cloning and analyzing two fragments of HMW-GS gene promoter in wheat

利用聚合酶链反应 PCR 技术从小偃6号中获得400bp左右的扩增产物,将其与pGEM-TEasy载体连接后转入大肠杆菌,经过筛选获得HMW-8-P和HMW-38-P两种类型克隆.序列分析表明:HMW-38-P包括了HMW-GS14基因上游启动子及信号肽对应编码区,而另一段 HMW-8-P 为一未知HMW-GS基因启动子区及信号肽对应的编码区.将两序列和GenBank中已知的35种HWM-GS基因启动子区序列进行多序列比对,最后获得HMW-GS启动子的系统发生树.通过系统发生树可以清晰地看出位于不同染色体上的不同亚基类型的HMW-GS基因的进化关系,并可确定HMW-8-P为Glu-D-1类型HMW-GS的启动子区.小偃6号中Glu-D-1类型的亚基为2亚基,所以HMW-6-P为2亚基启动子区序列.

PCR product about 400 bp was obtained from the wheat(XiaoYan 6).It was linked with pGEM-T easy vector and two types of positive clones HMW-38-P and HMW-8-P were identified. Sequencing and similarity analyzing results show HMW-38-P includes the partial promoter and sequence responding to signal peptide of HMW-GS14 gene. After compared them with 35 kinds of known promoter sequence of HMW-GS gene,a polygenetic tree was built. From the tree, the evolution relationship among different chromosomal loci's HMW-GS gene was characterized clearly. Based on the tree we can deduce that HMW-8-P is the partial promoter and partial 5' sequence of HMW-GS2 gene.