摘要
利用多聚酶链式反应（ＰＣＲ）对来源于一种生物杀虫剂（ｂａｃｕｌｏｖｉｒｕｓ）的几丁质酶基因进行体外扩增，并插入载体质粒ｐＲＯＫ２中，然后转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）ＸＬ１Ｂｌｕｅ。用重组质粒ｐＲＯＫ２ＤＮＡ转化植物转基因载体——土壤农杆菌（Ａｇｒｏｂａｃｔｅｒｉａｔｕｍｅｆａｃｉｅｎｓ）ＬＢＡ４４０４菌株。借助于土壤农杆菌侵染烟草叶圆片，将目的基因导入。通过组织培养诱导生芽和生根，获得烟草再生植株。利用含卡那霉素的培养基对再生烟株进行初步筛选。进一步ＰＣＲ和Ｗｅｓｔｅｒｎ印迹检测结果表明：转基因烟草中有几丁质酶基因和蛋白的表达。初步检测结果表明：转基因烟草具有较高的几丁质酶活性。
he production of enzyme capable of degrading the cell walls of invading phytopathogenic fungi is an important component of the defense response of plants.The baculovirus derived chitinase,which can dissolve fungal wall,was amplified by polymerase chain reaction(PCR),inserted into plasmid vector pROK2,and transferred into Escherichia coli XL1 Blue.The recombinant plasmid pROK2 DNA were mobilized into Agrobacteria tumefaciens LBA 4404 by using Freeze thaw for transformation method.With the help of infection of A.tumefaciens the chitinase were transferred into tobacco leaf disks.By tissue culture,shoots were induced.The induced shoots were regenerated roots in MS Medium containing kanamicin sulfate(50μg/ml)The results of PCR and western blot assays showed the chitinase gene was expressed in flue cured tobacco varieties CF 80,K326 and Xanthinc.The chitinse enzyme activity in transformed tobacco plants is higher than untransformed ones.
出处
《中国烟草科学》
CSCD
1997年第4期47-50,共4页
Chinese Tobacco Science
关键词
烟草
几丁质酶基因
表达
Tobacco
Chitinase
Gene
Expression
