两段杨树叶绿体DNA片段的克隆及叶绿体定点转化载体的构建  

Cloning of Two DNA Fragments from the Chloroplast Genome of Populus tomentosa and Construction of Its Site-directed Chloroplast Transformation Vectors

作　　者：周奕华[1] 石东乔[1] 侯丙凯[1] 肖宇红[1] 张中林[2] 张丽华[1] 沈桂芳[2] 胡赞民[1] 陈正华[1] 

机构地区：[1]中国科学院遗传研究所,北京100101 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《高技术通讯》2001年第9期6-12,共7页Chinese High Technology Letters

基　　金：国家自然科学基金资助项目 (3 9970 62 4)

摘　　要：利用PCR方法 ,从毛白杨 (PopulustomentosaC .)叶绿体基因组中克隆了 1.7kb和 1.6kb的相邻DNA片段 ,对其进行序列分析表明 ,扩增片段分别具有 176 6个和 16 0 1个核苷酸 ,前者包括 3’rps12 ,rps7基因的编码区及其边界序列 ,后者包含ndhB基因的第一外显子和内含子。本文还构建了杨树这两个片段的限制酶切图谱 ,并以这两个相邻片段为同源重组片段 ,分别将绿色荧光蛋白 (GFP)基因和苏云金芽孢杆菌杀虫蛋白 (Bt)基因 ,及其原核表达框插入其间 ,构建了杨树叶绿体定点转化载体pPZG和 pPZB。这两个特异性载体将定位整合到杨树叶绿体基因组中反向重复区的rps7和ndhB基因的间隔区 ,并高效表达GFP和Bt基因。迄今为止 ,本文所报道的内容在国内。kb and 1.6 kb DNA fragments were amplified from the chloroplast genome of Populus tomentosa. Sequencing analysis indicated that these two fragments were adjacent. The fragment 1 containing 1766 base pairs included the encoding regions of 3' rps 12 and rps 7 gene and their flanking sequences; and the fragment 2 with the length of 1601 bp contained the exon 1 and intron of ndh B gene. The restriction endonuclease map of the cloned DNAs was also established. Using the two cloned DNAs as the recombinant fragments, the poplar site directed chloroplast transformation vectors were constructed by inserting the green fluorescent protein ( GFP ) gene, Bacillus thuringiensis insecticidal crystal protein ( Bt ) gene, and their pronucleus reading frames between them, respectively. The specific plastid vectors named pPZG and pPZB, would integrate into the inverted repeat region (rps7/ndhB) of chloroplast genome of poplar, and express the GFP or Bt gene with high efficiency. Till now, this is the first report on plastid transformation in poplar.

关 键 词：杨树 叶绿体定点转化 绿化荧光蛋白基因 苏云金芽孢杆菌杀虫蛋白基因 植物基因工程 基因克隆 载体构建 遗传改良 

分 类 号：Q785[生物学—分子生物学] Q244