卷叶贝母MCT基因克隆与表达分析

Cloning and Expression Analysis of MCT Gene in Fritillaria cirrhosa D. Don

本研究是基于转录组测序结果,通过PCR技术克隆卷叶贝母(Fritillaria cirrhosa D. Don)中MEP途径的2-C-甲基-D-赤藓醇-4-磷酸胱氨酰转移酶基因(2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase, MCT)的开放阅读框(open reading frame, ORF)序列,运用生物信息学方法对该基因进行分析,预测其编码蛋白的结构与功能,并通过荧光定量PCR (qRT-PCR)检测FcMCT基因在野生鳞茎和愈伤组织中的表达情况,为后续卷叶贝母MCT基因功能研究打下基础。通过PCR技术,获得了900 bp的FcMCT ORF片段,编码299个氨基酸,并与NCBI上公布的木薯、橡胶树、向日葵等植物MCT蛋白的相似性达80%以上;通过SOPMA和SWISS-MODE网站预测的Fc MCT蛋白的二级、三级结构,可以看出FcMCT蛋白的主要结构元件是无规卷曲和α螺旋构成;通过qRT-PCR检测野生状态的卷叶贝母中根、茎、叶、鳞茎和诱导状态的愈伤组织中的FcMCT基因表达水平,发现在愈伤组织的表达水平最高。最终,我们认为FcMCT是甾类生物碱合成途径的关键酶,在贝母不同组织中的表达量不同,受激素组合诱导表达,FcMCT是一个具有生物学功能的蛋白质,为利用基因工程手段提高卷叶贝母中生物碱含量提供了理论依据。

In this study, Open Reading Frame （ORF） of the 2-C-methyl-D-erythritol-4-cystinetransferase gene of MEP in Fritillaria cirrhosa （FcMCT） was cloned via PCR method based on RNA-Seq. Some bioinformatic methods and softwares were used for the gene structure and function analysis of this gene. Real-time quantitative PCR （RT-qPCR） method was used to detect the expression of FcMCT gene in wild bulb and callus, aiming to lay the foundation for the further study on the function ofMCT gene in FritiUaria cirrhosa. PCR detection showed that the FcMCT ORF was 900 bp, encoding 299 amino acids, which had 80% similarity with the MCT protein from Monihot esculenta, Hevea brasiliensis, and Helianthus annuus published by NCBI. The prediction results of FcMCT protein secondary and tertiary structure through SOPMA and SWISS-MODE websites indicated that the FcMCT protein was mainly composed of α helix and random coil. The expression level of FcMCT gene in root, stem, leaf, bulb of wild Fritillaria cirrhosa and callus of induced Fritillaria cirrhosa was detected by qRT-PCR, the results of which showed that the highest expression level was in callus. In the end, we considered that FcMCT was a key enzyme in the biosynthesis pathway of steroid alkaloid. The expression of FcMCT was different in different tissues of FritiUaria cirrhosa and it was induced by hormonal combination. FcMCT was a biologically functional protein, which laid a theoretical foundation for the improvement of the alkaloid content in Fritillaria cirrhosa by using genetic engineering.