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当前位置: 首页 > 文章 > 美洲黑杨木质素合成关键基因的克隆及反义表达载体的构建 中南林业科技大学学报 2010,30 (11) 77-82
Position: Home > Articles > Cloning of lignin biosynthesis key genes and construction of antisense expression vector to Populus deltoides Journal of Central South University of Forestry & Technology 2010,30 (11) 77-82

美洲黑杨木质素合成关键基因的克隆及反义表达载体的构建

作  者：
曹阳;袁澍;徐飞;张中伟;薛立薇;林宏辉
单  位：
四川大学生命科学学院
关键词：
生物科学与技术;美洲黑杨;木质素合成;关键基因;克隆技术;反义表达载体
摘  要：
以美洲黑杨Populus deltoides新萌叶片为材料,通过自行设计引物,用RT—PCR的方法克隆了美洲黑杨木质素合成关键基因4CL和CAD基因部分序列。测序结果表明:这两个基因片段长度分别为859 bp和493 bp。此外,人工合成了4CL基因木质部特异表达启动子4CLp,长度为1 180 bp。分别用限制性内切酶Hind III/BamH I、BamHI/Sma I和Sma I/Sac I对4CLp、4CL和CAD基因序列进行双酶切,同时用Hind III/Sac I对pBI121表达载体进行双酶切,回收了目的片段。酶切回收后用T4DNA连接酶克隆到植物表达载体pBI121中,并转化至大肠杆菌感受态细胞DH5α。经质粒PCR和双酶切分析,确定获得了pBI—4CLp—a4CL—aCAD反义植物表达载体。
译  名：
Cloning of lignin biosynthesis key genes and construction of antisense expression vector to Populus deltoides
作  者：
CAO Yang,YUAN Shu,XU Fei,ZHANG Zhong-wei,XUE Li-wei,LIN Hong-hui(School of Life Science,Sichuan University,Chengdu 610064,Sichuan,China)
关键词：
bio-science and technology;Populus deltoides;lignin synthesis;key gene;clone technique;antisense expression vector
摘  要：
Populus deltoides leaves by indoor potted plant were used as tested materials to clone two lignin biosynthesis key genes(4-coumarate-CoA ligase(4CL) gene and Cinnamyl alcohol dehydrogenase(CAD)gene)by RT-PCR method and using designed primers.Sequencing results reveal that the cDNA lengths are 859 bp and 496 bp respectively.Additionally,xylem promoter sequence of 4CL gene(4CLp)was artificial synthesized,and the length is 1 180 bp.4CLp,4CL and CAD genes were cut by Hind III / BamH I,BamH I / Sma I and Sma I / Sac I respectively.Then the 4CL and CAD fragments were inserted to pBI121 vector reversely,whose 35S promoter was replaced by 4CLp.Therefore,the expression of these two antisense-sequences was controlled by 4CLp promoter,and the constructed vector is named pBI-4CLp-a4CL-aCAD.The vector was introduced into Escherichia coli DH5α.Plasmid PCR and restriction-enzyme double digestion further proved the antisense expression vector was successfully constructed.

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