果聚糖蔗糖转移酶基因的克隆及耐盐转基因烟草的培育  

The Cloning of Levansucrase Gene and Its Engineering of Salttolerant Tobacco Plants

作　　者：张慧[1] 董伟[1] 周骏马[1] 杜宝兴 谷冬梅[1] 陈受宜[1] 

机构地区：[1]中国科学院遗传研究所

出　　处：《生物工程学报》1998年第2期180-186,共7页Chinese Journal of Biotechnology

基　　金：国家高技术计划

摘　　要：采用ＰＣＲ方法克隆了枯草杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）果聚糖蔗糖转移酶基因（ＳａｃＢ），将其与克隆自酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）的羧肽酶Ａ的液泡引导信号序列连接得到嵌合基因。测序验证后，插入含ＮＰＴⅡ基因的植物双元表达载体ｐＢｉｎ４３８中，经农杆菌介导转化烟草。部分经卡那霉素筛选的抗性芽能在含１％ＮａＣｌ的ＭＳ培养基上正常生根，而未转化芽不能生根或根生长缓慢。转基因小苗移入盛蛭石的花盆并浇灌含１％ＮａＣｌ的ｈｏａｇｌａｎｄ′ｓ营养液，１７ｄ后，其中一些转基因烟草植株生长良好，而未转化苗出现明显萎蔫。ＰＣＲ扩增及Ｎｏｒｔｈｅｒｎ分析证实ＳａｃＢ基因已导入转基因植株并得到转录。此结果表明ＳａｃＢ基因的植物基因工程可提高烟草植株的耐盐性。SacB gene from Bacillus subtilis,which encodes levansucrase,was cloned by the PCR method and then coupled to the carboxypeptidase Y vacuolar sorting signal (cpy) from Saccharomyces cerevisiach.The chimeric gene was sequenced and inserted into the plant binary expression vector (pBin438) containing NPT Ⅱ gene,then transferred into tobacco (Nicotiana tabacum var.K326) by agrobacterium mediated transformation.Following selection by kanamycin,some transformed shoots were normally rooted on MS medium supplemented 1%NaCl,but the controls were not.The plantets were transferred to pots containing vermiculite and watered with hoagland’s nutrient solution added 1% NaCl and showed less growth inhibitation than controls.By PCR and Northern analysis,it was verified that the SacB gene had been integrated and transcripted in those transgenic plants.The results indicated that the SacB gene could enhance salt tolerance of transgenic plant.

关 键 词：果聚糖 蔗糖 转移酶基因 转基因 烟草 耐盐性 

分 类 号：Q555[生物学—生物化学] S572.033[农业科学—烟草工业]