安徽农学通报
安徽農學通報
안휘농학통보
AUHUI AGRICULTURAL SCIENCE BULLETIN
2015年
8期
13-14
,共2页
复制酶基因%载体构建%农杆菌%黄瓜花叶病毒
複製酶基因%載體構建%農桿菌%黃瓜花葉病毒
복제매기인%재체구건%농간균%황과화협병독
Replicase gene%Vector construction%Agrobacterium tumefaciens%Cucumber mosaic virus
用PCR方法扩增黄瓜花叶病毒部分复制酶基因(CMV△Rep),连接到PUCm-T载体上构建成克隆载体PUCm-T-CMV△Rep。用BamHⅠ和SalⅠ分别对克隆载体PUCm-T-CMV△Rep和植物表达载体pBIN438进行双酶切,获得目的片段和线性质粒。在T4 DNA连接酶的作用下进行定向连接,构建成植物表达载体pBIN438-CMV△Rep。采用CaCl2冻融法将重组子导入根癌农杆菌LBA4404。经PCR和双酶切鉴定,表明重组质粒pBIN438-CMV△Rep已成功导入根癌农杆菌中。
用PCR方法擴增黃瓜花葉病毒部分複製酶基因(CMV△Rep),連接到PUCm-T載體上構建成剋隆載體PUCm-T-CMV△Rep。用BamHⅠ和SalⅠ分彆對剋隆載體PUCm-T-CMV△Rep和植物錶達載體pBIN438進行雙酶切,穫得目的片段和線性質粒。在T4 DNA連接酶的作用下進行定嚮連接,構建成植物錶達載體pBIN438-CMV△Rep。採用CaCl2凍融法將重組子導入根癌農桿菌LBA4404。經PCR和雙酶切鑒定,錶明重組質粒pBIN438-CMV△Rep已成功導入根癌農桿菌中。
용PCR방법확증황과화협병독부분복제매기인(CMV△Rep),련접도PUCm-T재체상구건성극륭재체PUCm-T-CMV△Rep。용BamHⅠ화SalⅠ분별대극륭재체PUCm-T-CMV△Rep화식물표체재체pBIN438진행쌍매절,획득목적편단화선성질립。재T4 DNA련접매적작용하진행정향련접,구건성식물표체재체pBIN438-CMV△Rep。채용CaCl2동융법장중조자도입근암농간균LBA4404。경PCR화쌍매절감정,표명중조질립pBIN438-CMV△Rep이성공도입근암농간균중。
Cucumber mosaic virus partial replicase gene were amplified by PCR(CMV Rep),connected to the PUCm-T vector to construct the cloning vector of PUCm-T-CMV Rep. With BamH I and Sal I of PUCm-T-CMV cloning vector Rep and the plant expression vector pBIN438 were digested,obtained fragment and linear plas?mid. Directional connection in T4 DNA ligase,a plant expression vector was constructed by pBIN438-CMV Rep. Using CaCl2 freeze-thaw method the recombinant plasmid into Agrobacterium LBA4404. By PCR and double enzyme diges?tion showed that the recombinant plasmid,pBIN438-CMV Rep has been successfully introduced into Agrobacteri?um tumefaciens.