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

利用PCR方法，从毛白杨（Populus tomentosa C.）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1766个和1601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、外尚属首次。

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

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.