番茄γ-生育酚甲基转移酶基因的克隆与分析

Cloning and Sequence Analysis of γ-Tocopherol Methyltransferase (γ-TMT) Gene in Tomato

[目的]为进一步研究γ-生育酚甲基转移酶(TMT)基因的功能奠定基础。[方法]以拟南芥γ-TMT cDNA为信息探针,在GenBankdbEST数据库中搜索与其高度同源的番茄EST序列,通过人工序列拼接得到番茄γ-TMT的cDNA序列。据此拼接序列设计1对特异引物,以番茄叶片cDNA第1链产物为模板进行RT-PCR,回收目的片段,转化到大肠杆菌DH5α进行TA克隆。最后对获得的阳性克隆进行测序,分析测序结果与其他物种中该基因之间的同源性,进行氨基酸序列比对及系统进化分析。[结果]经RT-PCR扩增出1300bp左右的片段,成功克隆了番茄γ-TMT基因。克隆片段全长1354bp,包含1个1089bp的完整开放读码框,与拼接序列完全一致。番茄γ-TMT基因编码的氨基酸序列与马铃薯、小麦、玉米和拟南芥的γ-TMT基因的同源性分别达89%、75%、75%和70%。系统进化分析表明,番茄与马铃薯的γ-TMT基因有较近的亲缘关系。[结论]该试验中克隆的番茄γ-TMT基因编码的蛋白质分子量为39.8kD,等电点为8.28。

[Objective] The research aimed to lay foundation for further research on the function of γ-tocopherol methyltransferase（ TMT）gene. [Method] With the cDNA sequence of γ-TMT in A rabidopsis thaliana as querying probe,EST sequence with high homology were searched in dbEST database on GenBank website and cDNA sequence of tomato γ-TMT was obtained through artificial sequence splicing. According to this splicing sequence, a pair of specific primers was designed. With the first chain products of cDNA in tomato leaves as templates, RT-PCR was carried out. The target fragment was reclaimed and transformed into Escherichia coli DH5α for TA cloning. Finally the obtained positive clone was sequenced, the homology between the sequencing results and this gene in other species was analyzed and amino acid sequence alignments and the phylogenetic analysis were carried out. [Result]About 1 300 bp fragment was amplified through RT-PCR and γ-TMT gene in tomato was successfully cloned. The cloning fragment with the full length of 1354 bp contained a complete open reading frame （ORF） of 1 089 bp, which was completely accordant with the splicing sequence. The homologies between the amino acid sequences coded by γ-TMT gene in tomato and that of γ-TMT gene in potato, wheat, maize and A. thaliana were 89 %, 75 %, 75 % and 70 % respectively. The phylogenetic analysis showed γ-TMT between tomato and potato had closer genetic relationship. [Conclusion] The molecular weight of protein coded by γ-TMT gene in tomato that was cloned in the test was 39.8 kD and its isoelectric point was 8.28.