暴马桑黄MVD基因cDNA全长克隆及表达特性分析

The cloning and expression analysis of mevalonate pyrophosphate decarboxylase gene cDNA sequence from Sanghuangporus baumii

【目的】对暴马桑黄中参与三萜合成途径的关键酶——甲羟戊酸焦磷酸脱羧酶(MVD)基因进行克隆及表达特性分析,以了解暴马桑黄三萜合成的调控机制。【方法】利用生物信息学软件对MVD基因cDNA序列进行分析,并采取同源重组方法构建原核表达载体。用qRT-PCR技术分析MVD基因在暴马桑黄不同发育阶段表达特性;以分光光度计法测定暴马桑黄在不同发育阶段的三萜含量和变化规律。【结果】暴马桑黄MVD基因cDNA序列全长1209 bp,编码402个氨基酸,相对分子质量为43.43 ku,命名为SbMVD(登录号为MK977617)。系统进化树表明:暴马桑黄MVD蛋白和紫芝、灰树花MVD蛋白同源性最高。SDS-PAGE结果显示:在65 ku(包含21 ku标签蛋白)附近出现与预期大小一致的目的蛋白条带。qRT-PCR分析及三萜含量测定结果表明:暴马桑黄SbMVD基因转录水平和三萜含量在不同发育阶段呈先升后降的变化趋势,并且两者变化趋势基本一致。【结论】通过对SbMVD基因的克隆及表达特性分析,推测该基因在三萜合成途径中发挥一定作用,为进一步研究该基因在暴马桑黄三萜合成过程中的功能奠定了基础。

【Objective】To clone and characterize a mevalonate pyrophosphate decarboxylase(MVD)gene involved in the triterpenoids biosynthesis pathway in Sanghuangporus baumii is to understand the regulation mechanism of the triterpenoids biosynthesis pathway.【Method】The characteristics of the MVD gene sequence were determined using a series of bioinformatic tools.The prokaryotic expression vector was constructed using homologous recombination.Moreover,qRT-PCR was performed to measure the MVD gene transcript level,and spectrophotometry was used to determine the content and variation of triterpenoids at different developmental stages of S.baumii.【Result】The sequence analysis showed that the MVD gene cDNA sequence length was 1209 bp and encoded 402 amino acids.The molecular weight of the protein was predicted to be 43.43 ku,and it was named SbMVD(Gen Bank number MK977617).Amino acid sequence alignment revealed that the SbMVD protein shared the greatest homology with Ganoderma sinense and Grifola frondosa.SDS-PAGE electrophoresis showed that the target protein band was located at approximately 65 ku(including a 21 ku tag protein),which was consistent with the predicted protein.Furthermore,qRT-PCR and spectrophotometry results showed that the SbMVD gene transcript level and triterpenoids content increased before decreasing dynamically at different developmental stages.The variation trends were basically the same.【Conclusion】The cloning and analysis of the SbMVD gene indicated that it might play an important role in the triterpenoids biosynthesis pathway.Our results lay a foundation for further elucidating the triterpenoids biosynthesis function of the SbMVD gene in S.baumii.