毛白杨叶绿体基因启动子和终止子的克隆及其功能鉴定

摘 要：从毛白杨(Populus tomentosa)无性系植株组培苗中提取基因组总DNA，通过合成3对特异性引物，用PCR的方法扩增了包含psbA基因启动子、终止子和16S rRNA基因启动子的叶绿体DNA序列。用BLAST及DNAMAN对克隆序列进行了分析，并结合前人的研究工作，确定了两个启动子Prrn、PpsbA的-35区、-10区以及翻译调控序列等重要调控序列元件和终止子的“TAA”序列。并在序列分析的基础上合成新的引物，准确克隆了毛白杨的叶绿体16S rRNA基因启动子Prrn、psbA基因启动子PpsbA及其终止子TpsbA。利用叶绿体基因启动子的原核表达特性，分别用启动子Prrn（其后添加rbcL基因的RBS序列）和PpsbA及终止子TpsbA分别构建了GFP基因的原核表达载体pSGmT、pPGmT，并以T7启动子为对照构建了GFP基因的原核表达载体pETGm，进行了初步的原核表达实验，检测结果表明：克隆到的叶绿体基因启动子和终止子是有生物学功能的，在大肠杆菌BL21中呈组成型表达，同时还证明了RBS序列在基因表达中的重要性。

Cloning and Function Identification of the Promoters and Terminator of Populus tomentosa Chloroplast Genes

Using three pairs of specific primers, DNA fragments containing the promoter of 16S rRNA gene and the promoter and terminator of psbA gene, were obtained by PCR from Populus tomentosa chloroplast genomic DNA. Sequence analyzing results showed that the functional domains such as -35 domain, -10 domain and the translation control sequence motif in the two cloned promoters were conserved to Nicotiana tabacum. The promoter Prrn, PpsbA and the terminator TpsbA were cloned and the prokaryotic expression vectors of GFP were constructed by using Prrn with rbcL RBS sequence or RBSm sequence, PpsbA and T7 promoter, and the terminator TpsbA, respectively. Prokaryotic expression results showed that the promoters Prrn and PpsbA cloned were functional and their activities could be controlled by the RNA polymerase of E. coli BL21 and the GFP controlled by them could be constitutively expressed E.coli in BL21. T7 RNA polymerase could control them in a certain degree but weaker than controlling T7 promoter. It also showed that the RBS sequence was very important in gene expression when using Prrn. The expression of the target gene could be extremely influenced by the mutation of the RBS sequence.