油桐β-酮脂酰-CoA合成酶(KCS)基因的克隆与序列分析

Cloning and sequence analysis of β-ketoacyl-CoA synthase(KCS) gene in Vernicia fordii

为给油桐β-酮脂酰-CoA合成酶基因(KCS)结构和功能的研究提供参考,以油桐种子为材料,根据油桐转录组测序分析结果设计引物,利用RT-PCR技术克隆KCS基因,并进行了序列分析。获得了1条KCS基因的全长cDNA,命名为Vf_KCS。KCS基因的CDS长度为1 338 bp,编码446个氨基酸。该基因编码的蛋白质具有3个酶活性位点、6个产物结合位点、5个丙二酰-CoA结合位点和多个二聚体形成位点;Vf_KCS有2个明显的跨膜螺旋拓扑结构和3个疑似跨膜螺旋拓扑结构;二级结构主要由α-螺旋、β-折叠和不规则卷曲构成,该蛋白为亲水性蛋白。Vf_KCS编码的氨基酸序列与其它物种的KCS氨基酸序列具有较高的相似性,其中与葡萄的KCS基因相似度最高达89.69%,与其它植物的KCS基因所编码的氨基酸同源性大多在80%以上。进化树分析结果表明,Vf_KCS与拟南芥亲缘关系最为接近。预测分析结果表明,该酶有高度保守的催化区域。

In order to provide a reference for the studies on cloning and analyzing β-ketoacyl-CoA synthase gene in Verniciafordii, taking seeds in Efordii as materials, primers were designed according to Efordii transcription sequencing analysis results, KCS gene was cloned and analyzed by RT-PCR techniques. A full-length KCS cDNA was cloned and named by Vf_KCS. The CDS length of Vf_KCS gene is 1 338 bp, coding 446 amino acids. The protein has three active sites, six product binding sites, five malonyl-CoA binding sites and several dimer interfaces. Vf_KCS has two obvious transmembrane helix topology structures and three suspected transmembrane helix topology. The protein is hydrophilic protein which is mainly composed of a-helix, B-folded and random coil structures in the secondary structure. The sequence of Vf_KCS has high similarity with other species, Vf_KCS has the highest sim^ilarity with Vitis vinifera, up to 89.69%, and more than 80% with other plants. The results of phylogenetic analysis show that Vf_KCS has the closest relatives with Arabidopsis thaliana. The results of prediction and analysis indicate that the enzyme has highly conserved catalytic region.