切割桑花叶萎缩类病毒的12串联体核酶基因克隆与转录载体构建  

Cloning and Transcription Vector Construction of a 12 Multimeric Ribozyme Gene Targeting Mulberry Mosaic Dwarf Viroid

作　　者：孔卫青[1] 杨金宏[1] 

机构地区：[1]安康学院陕西省蚕桑重点实验室,陕西安康725000

出　　处：《蚕业科学》2013年第4期638-642,共5页ACTA SERICOLOGICA SINICA

基　　金：陕西省科技厅农业攻关项目(No.2011K01-40);陕西省教育厅重点实验室项目(No.11JS001)

摘　　要：通过人工合成和PCR扩增,克隆特异性切割桑花叶萎缩类病毒RNA的12串联体核酶基因,用于控制病毒基因的表达。将桑花叶萎缩类病毒正链RNA上10 nt的靶序列连接在锤头型核酶催化区3'端,构成具有自切割功能的核酶基因。克隆该核酶基因并将其连接于载体pMD18T-SP6。在SP6 RNA聚合酶的作用下对线性化的重组质粒进行体外转录,体外自切割反应显示转录的多体自切割核酶可以通过内部的顺式切割释放出核酶分子。同时,将该12串联体核酶基因导入双元质粒pBI121中,获得重组植物转录载体pBI121-12MRz,为进一步培育抗桑花叶型萎缩病的转基因桑树奠定了基础。Using artificial synthesis and PCR amplification methods,a 12 multimeric ribozyme gene targeting mulberry mosaic dwarf viroid was cloned for controlling the expression of viral genes.10 nt of sense RNA was chosen as the target sequence from mulberry mosaic dwarf viroid and ligated to the catalytic 3′ end of hammerhead ribozyme for construction of the self-cleavable ribozyme gene.Then,the ribozyme gene was cloned and ligated to vector pMD18T-SP6.The transcription of linearized recombinant plasmid was catalyzed by SP6 RNA polymerase in vitro,and the resultant multimeric self-cleaving ribozyme could release ribozyme molecules through internal cis-cleavage.Meanwhile,this 12 multimeric ribozyme gene was introduced into binary plasmid pBI121 to construct recombinant plant transcription vector pBI121-12MRz,establishing a good foundation for preparing transgenic mulberry resistant to mulberry mosaic dwarf disease.

关 键 词：桑花叶型萎缩病 类病毒 12串联体核酶基因 转录载体 切割活性 

分 类 号：S888.712[农业科学—特种经济动物饲养] Q782[农业科学—畜牧兽医]