摘要
用０１５％乙烯利诱导烟草幼苗后，提取叶片总ＲＮＡ，并通过反转录及多聚酶链式反应（ＲＴＰＣＲ）扩增出β１，３葡聚糖酶基因的ｃＤＮＡ。将其克隆至载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ后，完成了此基因的全序列分析。测序结果表明：所克隆的ｃＤＮＡ除第１００８位碱基与所报道的不同外，其它序列完全一致。为制备抗血清，构建了此基因的大肠杆菌表达载体，并在表达宿主菌ＢＬ２１（ＤＥ３）ｐｌｙｓＳ中得到表达，进行了Ｗｅｓｔｅｒｎｂｌｏｔ分析。同时，还构建了此基因的植物表达载体。
Total RNA of tobacco leaf was isolated after treatment of the plant with ethylene.Using this RNA as template,the cDNA of β 1,3 glucanase was amplified by reverse transcription and successive polymerase chain reaction (RT PCR).After the cDNA was cloned into pBluescript SK,the whole sequence was analysed and the result showed that the sequence of the β 1,3 glucanase cDNA was almost identical with that of the reported gene,except that the 1008th base changed from T to C which did not change the coded amino acid.The expression of this gene in E.coli produced a protein of about 39kDa,an expected size for β 1,3 glucanase,and Western blot of the expression product was analysed.Plant expression vector of this gene was constructed and plant transformation with this vector is in progress.
出处
《生物工程学报》
CAS
CSCD
北大核心
1998年第4期412-418,共7页
Chinese Journal of Biotechnology
基金
中国科学院植物生物技术实验室和国际科学与文化中心(ICSC)世界实验室资助
关键词
烟草
葡聚糖酶
cDNA
大肠杆菌
植物表达
载体
β 1
3 glucanase gene
sequencing
expression in E.coli
plant expression vector
