苦荞4-香豆酸辅酶A连接酶基因(Ft4CL)的克隆及序列分析

Cloning and sequence analysis of 4-coumarate: CoA ligase gene from Fagopyrum tatarium

关键词:  苦荞  4-香豆酸辅酶A连接酶  基因克隆  序列分析

Key words:  Fagopyrum tatarium  4-coumarate  CoA ligase  gene cloning  sequence analysis

摘要:

以苦荞栽培种‘西荞2号'为材料,利用同源克隆和RT-PCR技术获得Ft4CL保守片段,采用RACE技术获得Ft4CL基因的3'末端及5'末端序列,并进一步采用生物信息学方法进行序列分析。结果表明:从苦荞花蕾总RNA中获得一条苦荞麦(Fagopyrum tatarium)4-香豆酸辅酶A连接酶基因(4-coumarate: Coa ligase, Ft4CL)的cDNA全长序列。生物息学分析结果显示,Ft4CL基因ORF全长1 602 bp,可编码553个氨基酸,理论标准分子质量为58.02 kDa,等电点(pI)为5.23。该研究首次从苦荞中获得Ft4CL基因的cDNA全长序列,该基因具有植物4CL同源基因的典型特征,推导的氨基酸序列具有4CL的所有活性位点并归属于黄酮代谢支路。该研究结果可为深入研究苦荞黄酮代谢途径奠定基础,为采用代谢工程技术提高苦荞黄酮含量提供候选靶基因。

Abstract:

This study focused on cloning and characterizing the 4-coumarate: CoA ligase gene(Ft4CL)from Fagopyrum tatarium. Using buckwheat species ‘Xi Qiao No.2', according to the conserved squences of 4CL from GenBank,a pair of degenerate primer was designed and synthesized. Through RT-PCR(reverse transcription PCR)technique,the conserved fragment of Ft4CL was amplified from the total RNA of F. tataricum flower buds. Then,the RACE technique(rapid-amplification of cDNA ends)was performed,and the 5' end and 3' end of Ft4CL were succesfully amplified,respectively. The complete cDNA of Ft4CL was obtained by splicing the above sequences,and a pair of gene-specific premie was synthesized to amplify the ORF(open reading frame)regine of Ft4CL. Using DNAman software to deduce the ORF sequence of Ft4CL to the amino acid sequence,its homologous with other 4CLs were analyzed by NCBI Blast tool. The secondary structure of Ft4CL was predicted by SOPMA(http://pbil.ibcp.fr),multiple sequence alignment was performed by DNAman software, and phylogenetic tree was built with neighbor-joining method by MEGA 5.0. The results were as follows: the similarity of Ft4CL with F. esculentum(HM149785)showed the hightest level(up to 94%)and it ranging form 66%-75% with other plant 4CLs. Multiple sequence alignment results showed that the Ft4CL had conserved motifs of BOXⅠand BOXⅡ near by the C-terminus,but it had relatively low similarity with other 4CLs at the C-terminus. According to the phylogenetic tree analysis results,the selected 4CLs were grouped into 2 cluster,Ft4CL,Arabidopsis thaliana 4CL1,and A. thaliana At4CL2 belonged to ClusterⅠ. In conclusion, the results could provide basic data for in-depth study of Fagophrum tatarium flavonoid pathway. Furthermore,this study indicated that Ft4CL could be a new candidate target gene for developing high flavoniod F. tatarium by metabolic engineering technology in future.