酿酒酵母HAL1基因的克隆及植物表达载体的构建

CLONING OF HAL1 GENE FROM SCCHAROMYCES CEREVISIAE AS2.375 AND THE CONSTRUCTION OF PLANT EXPRESSION VECTOR OF HAL1 GENE

HAL1基因是酵母中重要的耐盐基因。以酿酒酵母AS2 .375菌株的DNA为模板 ,根据已发表的序列设计引物 ,经PCR扩增得到约 90 0bp的HAL1基因片段 ,连接到 pMD1 8 T载体上 ,转化大肠杆菌JM 1 0 9,筛选重组质粒进行酶切分析和序列测定 ,结果显示已克隆到完整的可读框 ,该基因的序列与已知序列同源性达 99%。将HAL1基因从T 载体上切下连接到pAM 1 94载体上构建了HAL1基因的植物表达载体 ,用于烟草的转化获得了耐盐性提高的转化植株。

HAL1 gene is an important salt tolerant gene in yest.Total genomic DNA was isolated from Sccharomyces cerevisiae AS2.375.An about 900 bp DNA fragment was obtained with PCR technique by using the primer that designed according to the published sequence.The DNA fragment was cloned to T vector and transformed to E.coli strain JM109.The restriction map of the recombinant plasmid was analyzed and the DNA fragment was sequenced.The result showed that the entire open reading frame had been cloned and the identity of it's sequence to the published sequence is more than 99%.After cutting the HAL1 fragment from the T vector,we ligated the fragment to the pAM194 vector and obtained the plant expression vector of HAL1 gene.The transformants of tobacco with improved salt tolerant have been screened.