青稞4-香豆酸辅酶A连接酶基因4CL的克隆及表达分析

Cloning and Expression Analysis of 4-Ceoumarate:CoA Ligase Gene 4CL in Hulless Barley

为了进一步了解4-香豆酸辅酶A连接酶(4-coumarate:CoA ligase;4CL)基因在黄酮合成中的作用,利用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)获得了青稞4CL基因(Hv4CL)的cDNA全长序列,并利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术,对该基因在青稞4个胚乳发育时间段茎、叶和花中的表达情况进行研究。结果表明,Hv4CL基因cDNA序列全长2 107bp,该序列包含一个完整的开放阅读框(ORF,1 662bp),编码553个氨基酸。Hv4CL与小麦同源性最高(96%),且该蛋白序列中包含2个保守的box,即AMP结合盒:boxⅠ(SSGTTGLPKGV)和boxⅡ(GEICIRG)。qRT-PCR分析结果表明:4CL基因在青稞不同发育时期、不同组织的表达丰度不同,在茎中表达量最高,其次是叶和籽粒。

In order to further understand the role of 4-coumarate:CoA ligase(4CL)gene in flavonoids biosynthesis of barley,the full length cDNA encoding the 4CL gene was cloned by RT-PCR and rapid amplification of cDNA ends(RACE)in Qingke(hulless barey,Hordeum vulgare L.var.nudum Hook.F)and its expression was analyzed in different tissues(stem,leaf and flower)during 4periods during endosperm development by quantitative real-time PCR(qRT-PCR).Sequence analysis indicated that the full-length cDNA of 4CL was 2 107 bp,contained 1 662 bp in open reading frame(ORF)which encoded 553 amino acid residues.The deduced amino acid sequence contained two conserved boxes of the AMP binding box:boxⅠ(SSGTTGLPKGV)and boxⅡ(GEICIRG),which was most similar to 4CLof wheat with 96%amino acid identity.The qRT-PCR results indicated that obvious differences in the expression level of the 4CL gene were existed in different tissues at different developmental stages,and the expression level in stem was predominant,while the least in seeds,indicating that its expressive models in different tissues and organs of plant were different.The study could layafoundation to raise the content of flavonoids in barley by regulating the expression of 4CL gene and provide reference to further investigate the expression models of 4CL gene in different tissues and organs of plant.