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BcMF3反义基因植物表达载体的构建及其对拟南芥的遗传转化
引用本文:刘乐承,余小林,叶纨芝,向珣,卢钢,曹家树.BcMF3反义基因植物表达载体的构建及其对拟南芥的遗传转化[J].长江大学学报,2005,2(11):76-79.
作者姓名:刘乐承  余小林  叶纨芝  向珣  卢钢  曹家树
作者单位:浙江大学蔬菜研究所,浙江,杭州,310029;长江大学园艺园林学院,湖北,荆州,434025,浙江大学蔬菜研究所,浙江,杭州,310029
基金项目:中国科学院资助项目 , 浙江省科研项目
摘    要:根据编码白菜(Brassica rapa L. ssp. chinensis,syn B. campestris ssp. chinensis)果胶甲酯酶的BcMF3 基因cDNA 序列内部第1 343~1 797个碱基之间序列设计引物,从白菜花蕾cDNA中PCR扩增出455 bp的片段,把该片段作为反义基因连接至双元载体pBI12l上,得到了植物表达栽体pBI-B3,并用"三亲杂交"法导入农杆菌LBA4404菌株中,PCR扩增和酶切结果表明所构建的植物表达载体pBI-B3含有BcMF3 反义基因片断,并已导入了根癌农杆菌(Agrobacterinm tumefuciens)中;采用花序浸润法转化拟南芥(Arabidopsis thaliana)后,播种筛选浸润植株的种子获得了5 株卡那霉素抗性植株,PCR 扩增证明BcMF3反义片段成功整合到拟南芥基因组中;转基因拟南芥子代(T2)的抗性分离符合孟德尔分离比例,表明外源基因是单一位点插入.

关 键 词:反义基因  拟南芥(Arabidopsis  thaliana)  BcMF3  白菜  表达载体  遗传转化  基因植物  表达载体的构建  拟南芥基因组  遗传转化  Arabidopsis  Thaliana  Genetic  Transformation  Antisense  Containing  Plant  Expression  Vector  点插入  外源基因  分离比例  孟德尔  抗性植株  转基因  整合  卡那霉素  种子  筛选  播种
文章编号:1673-1409(2005)11-0076-04
修稿时间:2005年9月26日

Construction of Plant Expression Vector Containing the Antisense BcMF3 and Its Genetic Transformation of Arabidopsis Thaliana
LIU Le-cheng,YU Xiao-lin,YE Wan-zhi,XIANG Xun,LU Gang,CAO Jia-shu.Construction of Plant Expression Vector Containing the Antisense BcMF3 and Its Genetic Transformation of Arabidopsis Thaliana[J].Journal of Yangtze University,2005,2(11):76-79.
Authors:LIU Le-cheng  YU Xiao-lin  YE Wan-zhi  XIANG Xun  LU Gang  CAO Jia-shu
Abstract:A 455 bp antisense gene fragment was amplified from nucleotides 1343 to 1797 of the cDNA sequence of BcMF3 gene that encodes a Pectin Methylesterase in Chinese cabbage-pak-choi.The produced fragment was introduced to the binary vector pBI121 to produce pBI-B3,and pBI-B3 was mobilized into Agrobacterium tumefaciens strain LBA4404 by triparental mating.PCR amplification and restriction digestion showed that the anti-gene fragment of BcMF3 was introduced into pBI121 and the generated pBI-B3 was transferred into the Agrobacterium tumefaciens successfully.Transformation of the A.tumefaciens strain LBA4404 harboring pBI-B3 to Arabidopsis thaliana was performed by floral dip,and 5 transgenic plants were obtained by kanamycin selection and confirmed by PCR amplification.The inheritance of selective marker gene transferred was in the model of the simple Mendel's fashion in progenies of the self-crossed generation,indicating that a single copy of the foreign gene had integrated into Arabidopsis thaliana genome.
Keywords:antisense gene  Arabidopsis thaliana  BcMF3  Chinese cabbage-pak-choi  expression vector  genetic transformation
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