辣椒中辣椒素生物合成途径基因Kas克隆及表达研究

Cloning and expression of Kas gene related to capsaicin biosynthesis in Capsicum annuum L.

3-氧酰基[酰基载体蛋白]还原酶(3-oxoacyl-[acyl-carrier-protein]synthase,Kas)在辣椒素生物合成途径中起着重要作用,但其全长DNA序列仍未见报道。以辣椒益都红基因组为模板,根据同源性设计特异引物PCR扩增得到辣椒Kas基因DNA序列(GenBank登录号:HQ229922)。Kas DNA序列全长3 456 bp,有8个外显子区域,编码488个氨基酸;推测蛋白分子质量为52.2 ku,等电点8.24。经同源比对分析得出,辣椒Kas与其他物种Kas有一定同源性;其中,与小米椒的遗传距离为0.0723。通过半定量RT-PCR分析发现,Kas在胎座中的表达量要高于肉质体中的表达量,授粉10 d后Kas的表达量逐渐降低。另外,植物激素油菜素内酯(Brassinosteroid,BR)对辣椒胎座Kas基因的表达有促进作用,在处理24 h后Kas表达量达到最高,随后逐渐降低。该基因的克隆与表达分析为深入研究辣椒Kas基因表达调控和辣椒素的生物合成机制奠定了重要基础。

A full length of 3 456 bp Kas （3-oxoacyl-[acyl-carrier-protein] synthase） gene （GenBank accession： HQ229922） was obtained from genomic DNA of Capsicum annuum Yidu-Red by PCR method. This gene consists of eight extrons, encodes 488 amino acids. The molecular weight and pl of the putative protein were 52.2 ku and 8.24, respectively. The putative amino acid sequences shared high identity to Kas gene of other plants. Especially Capsicum chinese, they share about 98% homology. In this study, the expression pattern of Kas gene at different time point after pollination in flesh and placenta was examined by semi-quantity RT-PCR. The mRNA abundance of Kas in placenta was higher than that of flesh. In addition, the Kas expression level decreased 10 d after pollination. Furthermore, the effect of brassinosteroid on this gene expression was investigated. The results showed that the mRNA abundance of Kas gene dramatically increased 6 h after treatment with brassinosteroid and reached a peak after 24 h and then decreased slowly. This results illustrated that brassinosteroid had a positive effect on Kas expression. Accordingly, this study will provide the basis for further studying the expression and regulation mechanism of Kas and its role in the biosynthesis mechanism of capsaicin.