一个特殊的顺式发夹核酶对感染烟草原生质体的影响  

A Specific Cis-Hairpin Ribozyme Facilitates the Infection in Tobacco Protoplasts

作　　者：王慧[1] 林志新[1] 孙庆申[1] 吴立刚[2] 范济华[2] 许政 

机构地区：[1]上海交通大学生命科学技术学院,上海200030 [2]纽约大学医学院,纽约10016 [3]中国科学院植物生理生态研究所,上海200052

出　　处：《上海交通大学学报》2004年第5期810-814,818,共6页Journal of Shanghai Jiaotong University

摘　　要：研究一个烟草花叶病毒(TMV)cDNA3′末端被特异切割的发夹核酶对感染原生质体的作用.选用一个外源基因绿色水母荧光蛋白(GFPuv)作为报道基因,取代了大部分的CP编码区域,以研究在烟草原生质体中发夹核酶对TMV载体的复制,通过GFP的表达来确定表达量.顺式发夹核酶对TMV载体感染效率的影响通过体外转录来观察.TMVGFPRIB体外转录感染原生质体的效率比与其不含该核酶的对应物的感染效率高3～5倍.Northern杂交结果表明,包括核酶在内的3′末端非病毒序列在体外转录反应开始后就被顺式发夹核酶切除了.当直接用载体DNApST-MVGFPRIB感染原生质体时,感染原生质体的数量比与之相对应的pSTMVGFPNOS感染原生质体数多50%～80%.This paper investigated the effect of a specific cis -hairpin ribozyme on a TMV -based vector TMV GFPRIB in the infection of tobacco protoplasts. The cis -hairpin ribozyme was designed to specifically cleave the TMV GFPRIB RNA at the site immediately downstream to the 3′terminus of the TMV RNA. The ribozyme was placed between the 3′terminus of the TMV genome and a polyadenylation signal sequence from NOS gene. The open reading frame for the coat protein (CP) gene in this vector was replaced with the GFPuv(green fluorescent protein) gene that allowed estimating the infection of the protoplasts by direct fluorescent microscopic observation. The effects of the cis -hairpin ribozyme on the infectivity of the TMV -based vector were observed as the in vitro transcripts. The in vitro transcript of TMV GFPRIB infected the protoplasts with the infection efficiency about 3 to 5 folds higher than its counterpart lacking the cis -hairpin ribozyme. Northern blot analysis indicates that the 3′terminal non-viral sequence including the ribozyme had been cleaved by the cis -hairpin ribozyme from the in vitro transcripts soon after the in vitro transcription reaction. When directly transfected the protoplasts with the vector DNA, pS TMV GFPRIB infected about 50% to 80% more protoplasts than its counterpart pS TMV GFPNOS did.

关 键 词：烟草花叶病毒 发夹核酶 绿色水母荧光蛋白 烟草原生质体 转染 

分 类 号：Q557.9[生物学—生物化学]