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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。
Gene-specific primers were designed according to the cDNA sequence of common wheat HKT1 (GenBank accession number: U16709), and the monoclonal BAC containing the HKT1 genomic sequence was obtained by screening the dwarf Chinese spring BAC library. According to the known sequence of HKT1 5’-end, the sequencing primers were designed and sequenced. The sequence was sequenced by using the BAC plasmid as a template. The sequence of 4 192 bp upstream of the start codon of HKT1 gene was obtained by Sequencer software. The sequence was analyzed using the Neural Network Promoter Prediction (NNPP) software, which predicted nine transcriptional start points. PLACE software analysis showed that the sequence has the basic elements of promoter TATA-box, CAAT-box, contains multiple stress inducing elements, such as salt-inducing element GAAAAA, antifreeze, water shortage, abscisic acid, cold tolerance components CANNTG and CCGAC , Injury inducing element TGACY, tissue specific expression element ACTTTA and ATATT. HKT1 promoter clones show that the method of gene promoter sequence selection using BAC library with gene-specific primers for direct sequencing using the BAC plasmid as a template is a practical method for cloning the gene promoter from the genome of common wheat.