暴马桑黄羊毛甾醇14α-脱甲基酶基因的克隆、序列分析及原核表达

Cloning,Sequence Analysis and Prokaryotic Expression of Lanosterol 14-alpha-demethylasegene from Sanghuangporus baumii

为深入了解暴马桑黄三萜合成途径的调控机理,基于前期测得的转录组数据,同时结合基因组数据(GenBank登录号为LNZH00000000.2),筛选得到暴马桑黄羊毛甾醇14α-脱甲基酶(lanosterol 14-alpha-demethylase,LSD)基因序列,并设计特异引物。以暴马桑黄菌丝体总RNA反转录得到的cDNA为模板,采用PCR技术克隆得到LSD基因cDNA序列全长,命名为SbLSD1(登录号为MN640689)。序列分析结果表明,SbLSD1基因cDNA序列全长1 746 bp,编码581个氨基酸,分子量为65.96 ku,没有信号肽,在53~75位氨基酸序列之间含有一个跨膜螺旋结构,属于P450超家族成员。系统发育分析表明,暴马桑黄LSD1蛋白和灵芝LSD蛋白同源性最高。将SbLSD1基因cDNA序列构建到原核表达载体pET-32a上,获得重组质粒pET-32a-SbLSD1,然后将其转入Escherichia coliBL21中进行SbLSD1蛋白诱导表达。SDS-PAGE结果显示,SbLSD1基因成功表达出蛋白,并在63~75 ku出现与预期大小一致的蛋白条带。通过对SbLSD1基因的克隆及表达分析,为进一步研究该基因在暴马桑黄三萜生物合成过程中的功能奠定基础。

In order to understand the regulation mechanism of triterpenoids biosynthesis pathway in Sanghuangporus baumii,combined with the genomic data of S.baumii from NCBI and the transcriptome data obtained in the early stage,the sequence of lanosterol 14-alpha-demethylase gene was screened out and specific primers were designed.The full length of the cDNA sequence of lanosterol 14-alpha-demethylase gene was cloned by PCR and named as SbLSD1(GenBank number MN640689).Sequence analysis showed that the total length of cDNA sequence was 1 746 bp,encoding 581 amino acid.The molecular weight of the protein was predicted to be 65.96 ku,there was no signal peptide,but the amino acid sequence 53-75 contained a transmembrane helix structure which belonged to P450 superfamily.Sequence alignment of amino acids revealed that SbLSD1 protein had the highest homology with Ganoderma lucidum LSD protein.Then,the sequence was constructed on the prokaryotic expression vector pET-32 a,and the positive plasmid pET-32 a-SbLSD1 was transferred into E.coli BL21.SDS-PAGE results showed that the protein was successfully expressed and the target protein band was located between 63-75 ku,which was consistent with the predicted protein.The cloning and expression analysis of SbLSD1 gene lay a foundation for further revealing the triterpenoids biosynthesis function of the SbLSD1 gene in S.baumii.