小麦高亲和性钾转运蛋白基因HKT1启动子克隆及序列分析

Cloning and Sequence Analysis of HKT1 Promoter in Wheat

根据普通小麦HKT1 cDNA序列(GenBank登录号:U16709)设计基因特异引物,通过筛选矮败中国春BAC文库,获得含有HKT1基因组序列单克隆BAC。根据HKT1 5'-端已知序列设计测序引物,以单克隆BAC质粒为模板进行测序,序列经Sequencer软件拼接,获得了HKT1基因起始密码子上游4 192 bp序列。用Neural Network PromoterPrediction(NNPP)软件分析此序列,预测存在9个转录起始点。PLACE软件分析表明,该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如盐诱导元件GAAAAA,抗冻、缺水、脱落酸、抗寒元件CANNTG和CCGAC等,伤害诱导元件TGACY,组织特异表达元件ACTTTA和ATATT等。HKT1启动子克隆表明,设计基因特异引物筛选BAC文库,通过以单克隆BAC质粒为模板直接测序获得基因启动子序列的方法是从普通小麦基因组中克隆基因启动子的一条切实可行的途径。

Using specific primers based on wheat HKT1 cDNA sequence（GenBank accession：U16709）,single BAC clone containing HKT1 genomic sequence was identified by screening Aibai Chinese Spring BAC library.4 192 bp HKT1 5′-flanking region was isolated by sequencing BAC plasmid.Nine transcription start sites were predicted by Neural Network Promoter Prediction（NNPP）program analysis.The functional elements were analysed by PLACE program.HKT1 promoter region contains the basic elements：TATA-box,CAAT-box,and stress-induced elements：salt-response element,cold-,dehydration-,ABA-and frozen-response elements,WUN-response elements and tissue-specific elements.Cloning of HKT1 promoter showed that it is a feasible method to isolate gene promoter by screening BAC library and sequencing BAC plasmid in wheat.