油桐β-酮脂酰-ACP还原酶(KAR)基因的克隆与序列分析

Isolation and Bioinformatics Analysis of β-ketoacyl-ACP Reductase (KAR) from Vernicia fordii

以葡萄桐近成熟种子为材料,根据油桐转录组测序分析结果设计引物,利用RT-PCR技术克隆获得了2条KAR基因的全长cDNA,分别命名为Vf_KAR1和Vf_KAR2(GenBank登录号:KF025646,KF025647)。生物信息学分析结果表明,Vf_KAR1和Vf_KAR2基因的CDS长度分别为993 bp和900 bp,分别编码331个和300个氨基酸;与其它物种的KAR氨基酸序列具有较高的相似性,Vf_KAR1与蓖麻的相似度最高,达82%;Vf_KAR2基因与花生、辣椒的相似度高达76%;2条KAR基因的多肽都含叶绿体转运肽,但是都没有信号肽;Vf_KAR1有1个明显的跨膜螺旋拓扑结构和1个疑似跨膜螺旋拓扑结构,Vf_KAR2有2个疑似的跨膜螺旋拓扑结构;肽链都表现为疏水性,都有典型的活化中心三联体(Ser-Tyr-Lys);三级结构都有典型的Rossmann折叠特征。因此,初步推测Vf_KAR1和Vf_KAR2是油桐β-酮脂酰-ACP还原酶KAR基因家族的两个成员。

Primers were designed according to the results of the transcriptome sequencing analysis of Vernicia fordii,two full-lengths KAR cDNA were cloned by RT-PCR technique from nearly ripe seed in Vernicia fordii,which were respectively named Vf_KAR1 and Vf_KAR2 （GenBank accession number：KF025646,KF025647）.The bioinformatics analysis showed that the CDS lengths of Vf_KAR1 and Vf_KAR2 genes were 993 bp and 900 bp respectively,coding 331 and 300 amino acids;the two sequences had high similarity with other species,Vf_KAR1 had the highest similarity with Ricinus communis,up to 82％ ;Vf_KAR2 had the highest similarity with Arachis hypogaea and Capsicum annuum,up to 76％ ;two different sequences of KAR contained chloroplast transit peptides,but no signal peptide; Vf_KAR1 had an obvious transmembrane helix topology structure and a suspected transmembrane helix topology,but Vf_KAR2 had only two suspected transmembrane helix topology;two polypeptide chains of KAR had hydrophilicity and typical active sites（Ser-Tyr-Lys） ;the predicted tertiary structure template of Vf_KAR and Vf_KAR2 also had typical characteristic with Rossmann fold.Therefore,the initial prediction was that Vf_KAR1 and Vf_KAR2 were two members of β-ketoacyl-ACP reductase gene family in Vernicia fordii.