棉花4-香豆酸辅酶A连接酶基因克隆及原核表达

本研究从棉花中克隆了一个4CL基因,命名为Gh4CL2(GenBank登录号为FJ848870)。研究结果表明:Gh4CL2基因cDNA全长2332bp,具有一个1725bp的开放阅读框,5′非编码区为64bp,3′非编码区为543bp,编码574个氨基酸,预测分子量约为62.106kD,等电点为5.94。氨基酸同源性分析发现,Gh4CL2与来自白杨、大豆和紫草的4CL一致性较高。进一步研究Gh4CL2基因的功能,构建了该基因的原核表达载体pET-28a-4CL2,经酶切鉴定后转化到大肠杆菌BL21(DE3)中。SDS-PAGE分析表明,最佳诱导表达条件为0.5mmol/LIPTG在37℃下诱导4h,重组蛋白主要以包涵体形式出现。

Cloning and Prokaryotic Expression of Gh4CL2 Gene from Gossypium hirsutum L.

4-coumarate:CoA ligase (4CL) is a key enzyme in the pathway of phenylpropanoid metabolism during lignin forming.A gene coding for 4CL,designated as Gh4CL2 (GenBank accession no.FJ848870) was isolated from cotton (Gossypium hirsutum L.).The full length Gh4CL2 cDNA is 2 332 bp,including a 64 bp 5'-UTR,an ORF of 1 725 bp,and a 543 bp 3'-UTR.This cDNA sequence encoded a polypeptide of 574 amino acid residues with a predicted molecular mass of 62.106 kD and a basic isoelectric point of 5.94.The deduced amino acid sequence had a high homology with 4CL from Populus tremuloides,Glycine max and Lithospermum erythrorhizon.To investigate the function of the Gh4CL2 gene,the full-length open reading frame was fused into a prokaryotic expression vector pET-28a.Double endonucleases digestion showed that the recombinant vector pET-28a-4CL2 was successfully constructed and transformed into E.coli BL21(DE3) cells.SDS-PAGE indicated that the most high expression quantity was induced by 0.5 mmol/L IPTG treatment for 4 h at 37℃,but the recombinant proteins mainly appeared as inclusion bodies.