蜡梅花香基因SAMT的cDNA克隆及在大肠杆菌中的表达

[目的]研究蜡梅花香基因SAMT cDNA的克隆及表达。[方法]以蜡梅花瓣总RNA为模板进行RT-PCR,扩增得到与预期大小相同的PCR产物带,回收RT-PCR产物,将其与PMD18-T载体进行T-A克隆连接并导入大肠杆菌DH5α,经菌落PCR和双酶切鉴定,筛选带有目的基因的重组质粒并进行测序。[结果]测序证实成功克隆得到蜡梅花香基因SAMT cDNA的ORF片段,其长度为1 196 bp,编码380个氨基酸残基,与已报导的蜡梅SAMT(ABU88887)的同源性为99.2%。将SAMT基因亚克隆到原核表达载体PGEX4T-1中获得重组菌种命名为PGSAMT,用0.01 mol/L的IPTG进行诱导表达,经SDS-PAGE分析,SAMT的融合表达蛋白分子量约为66 kDa,与预期的26 kDa的GST带和42.3 kDa的蜡梅SAMT基因编码蛋白构成的融合蛋白大小接近。[结论]该研究成功克隆并表达蜡梅花香基因SAMT。

cDNA Cloning and Expression of SAMT Gene from Chimonanthus praecox in Escherichia coli

[Objective] The aim was to study the cDNA cloning and expression of SMAT gene from Chimonanthus praecox.[Method] A novel cDNA was cloned by PT-PCR using the total RNA as template,and amplified to the desirable size.The RT-PCR products were reclaimed and transformed into E.coli DH 5ɑ together with the PMD18-T vector after ligating by T-A cloning.Identified by colony PCR and EcoRI and NotI digestion,the recombinant plasmid with target gene was screened out and conducted the sequence analysis.[Result] Results of the sequence analysis showed that the ORF fragment of the SAMT cDNA was successfully cloned form Chimonanthus praecox gene,with the length of 1 196 bp and encoded 380 amino acids fragment which shared 99.2% homology to that of previously reported SAMT cDNA from Chimonanthus praecox(ABU88887).The SAMT cDNA fragment was sub-cloned into the prokaryotic expression vector PGEX1-4T-1,and the obtained recombinant plasmid was named PGSAMT.After inducting by 0.01mol/L IPIG,the result of the SDS-PAGE analysis showed that the molecular weight of the fusion expression SAMT protein was about 66 kDa,which was close to the predicted fusion protein derived from the 26 kDa GST band and 42.3 kDa SAMT gene of Chimonanthus praecox encoded protein.[Conclusion] This study successfully cloned and expressed the SAMT gene of Chimonanthus praecox.