摘要
根据已报道的邻苯二酚 1 ,2 双加氧酶基因 (tfdC)序列 ,设计PCR引物 ,从一株邻单胞菌 (Plesiomonas)的 pL1质粒上扩增到tfdC基因片段 ,连接到 pGEM T载体上 ,并转化大肠杆菌JM1 0 9菌株 ,筛选到阳性克隆。序列分析结果表明 ,PCR产物全长 80 1bp ,有一个阅读框 ,编码 2 55个氨基酸 ,与增氧产碱菌 (Alcaligeneseutroplus)的tfdC基因相比 ,在 693位相差一个碱基 (C→A) ,导致编码产物在 2 2 8位相差一个氨基酸。将目的片段克隆到 pBluescrip tIIKS质粒载体上 ,筛选到阳性克隆 pBt1 2G ,在大肠杆菌JM1 0 9中可表达邻苯二酚 1 ,2 双加氧酶 (C1 2 0 )的活性 ;将翻译起始密码子为ATG的目的片段克隆到 pET 30a质粒载体上 ,筛选到阳性克隆 pET30A ,在大肠杆菌BL2 1 (DE3) plysS中可表达目的蛋白。C1 2 0是芳香族化合物降解过程中的关键酶 ,为了进一步在草坪草中表达tfdC基因 ,利用草坪草降解环境中芳香族污染物 ,将该基因翻译起始密码子由GTG突变成ATG ,突变后的基因在大肠杆菌中可正常表达C1 2 0的活性。
A new catechol\|1,2\|dioxygenase gene (\%tfd\% C) was cloned from the \%Plesiomonas\% using the PCR method. Primers were designed according to the reported sequence of Catechol\|1,2\|dioxygenase (C120) gene from \%Alcaligenes eutroplus\%. The amplified fragment contained a 765 bp open reading frame (ORF), encoding a protein of 255 amino acids. The new \%tfd\% C gene shared a high homology with the one cloned from \%Alcaligenes eutroplus\%, showing only one base difference at 693 site (C→A) and consequently one amino acid difference at 228 site (P→T). The ORF was cloned to the plasmid pBluescriptII KS, which was transferred to \%E.coli\% JM109 and a positive clone, pBt2G, was then selected. A significant activity of C120 was detected in the positive clone. When the ORF was cloned to the plasmid pET\|30a, which was transferred to \%E.coli\% BL21(DE3) plysS, the expected 33 kD protein was detected from a positive clone, pET30A, by SDS\|PAGE. The C120 is a key enzyme in degrading aromatic pollutants in the environment. In order to use plants to degrade aromatic pollutants, the gene will be introduced into the turfgrass. To express the gene properly in plants, its translation initiation codon was modified from GTG to ATG. A similar activity of C120 was obtained following the modification.
出处
《微生物学报》
CSCD
北大核心
2000年第6期579-585,共7页
Acta Microbiologica Sinica
基金
国家自然科学基金资助项目!( 3 9770 4 78)&&
关键词
邻单胞菌
(tfd
C)
PCR
克隆
表达
Plesiomonas
Catechol 1
2\|dioxygenase gene (\%tfd\% C)
PCR
Cloning
Expression
