罗汉果SgHMGR基因的克隆、分析及原核表达

Cloning,bioinformatics analysis and prokaryotic expression of SgHMGR in Siraitia grosvenorii

关键词:  罗汉果  3-羟基-3-甲基戊二酸单酰辅酶A还原酶  克隆  原核表达

Key words:  Siraitia grosvenorii  HMG-CoA Reductase  cloning  prokaryotic expression

摘要:

罗汉果甜苷V是一种葫芦烷型四环三萜类物质,作为主要的活性成分和甜味成分存在于成熟果实中,3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR)作为萜类化合物生物合成途径中的第一个限速酶,位于甲羟戊酸(MVA)途径中,是罗汉果甜苷V生物合成途径中的重要调控位点。为了深入了解罗汉果甜苷V的生物合成途径,该研究从罗汉果转录组数据中获得一条编码HMGR的unigene,以授粉后3 d的幼果作为实验材料,通过RACE技术获得了1 926 bp的全长序列,经过生物信息学软件分析,发现该基因含有1 749 bp的开放阅读框,编码582 氨基酸残基,含2段跨膜区,分别位于50～72 aa和93～115 aa,亚细胞定位预测位于质膜或内质网上,预测该蛋白没有信号肽,系统进化树分析显示与同科植物黄瓜和甜瓜中HMGR基因的同源性最高。该研究采取去掉N端跨膜区的方法,构建原核表达载体转化大肠杆菌BL21(DE3),经IPTG诱导在上清和沉淀中均有融合蛋白出现,尤其在25 ℃诱导过夜后上清中表达最明显。该文是首次对SgHMGR基因全长序列的克隆及原核表达的功能验证,为进一步深化SgHMGR基因在罗汉果甜苷V生物合成途径中的功能及分子调控研究打下基础。

Abstract:

Siraitia grosvenorii, belonging to Cucurbitaceae, is an herbaceous perennial plant native to South China and north Thailand, and mostly prevalent in Guilin City of Guangxi Zhuang Autonomous Region. The fruits are widely used as Chinese traditional medicine and meanwhile they contain extremely sweet flesh. As the main active component and sweetner ingredient, mogroside V is a kind of cucurbitane type tetracyclic triterpenes which only exists in the ripe fruits of S. grosvenorii. In the proposed mogoroside V biosynthetic pathway, there are two independent pathways for triterpenoid biosynthesis(MVA pathway and MEP pathway). 3-Hydroxy-3-methylglutaryl coenzyme-A reductase(HMGR), the first rate-limiting enzyme in mevalonate(MVA)pathway of terpenes biosynthesis, is the important regulatory site in mogroside V biosynthetic pathway in S. grosvenorii. However, there is little knowledge about function study of key genes involved in this pathway. In order to further understand mogroside V biosynthetic pathway, the full-length of SgHMGR was obtained by RACE-PCR method from 3 d after fertilization(DAF)of S. grosvenorii fruits based on the unigene of HMGR in transcriptome data, and further conducted by bioinformatic analysis. The recombinant prokaryotic vector was constructed using pET-32a and then transformed into Escherichia coli BL21(DE3)for expression. The results showed that a full-length SgHMGR cDNA was cloned with 1 927 bp and it contained 1 749 bp open reading frame(ORF)encoding a protein of 582 amino acids(aa)(GenBank No.HQ128556.1). The theoretical molecular weight(MW)and isoelectric point(PI)of this predicted protein were 62.6 kD and 8.18, respectively. The predicted protein contained the conserved domain of HMGR and proved it to be a member of HMGR family. SgHMGR protein had the high homology with Cucumis sativus and Cucumis melo of Cucurbitaceae plants, which were both 88%. Its subcellular localization was predicted in plasma membrane or endoplasm. By HMGR gene structure prediction analysis, the predicted SgHMGR protein had two transmembrane domains in N-terminal which were located in 50-72 aa and 93-115 aa, respectively. Besides, there were no predicted signal peptides for SgHMGR protein. In order to avoid the influence of transmembrane domains on the heterologous expression, SgHMGR from 116 amino acids with no transmembrane domains was cloned and named as ‘SgHMGR-1' in this study. The MW and PI of SgHMGR-1 were 49.6 kD and 5.95. The recombined vector was transformed into E. coli BL21(DE3)and expressed by IPTG. The prediction indicated that the MW and PI of the recombined protein of SgHMGR-1 and His were 65.8 kD and 5.95. The results of SDS-PAGE and Western blotting demonstrated that the fusion protein could be expressed in both supernatant and pellet after induction by IPTG overnight and it had highest expression in supernatant at 25 °C. This is the first report about cloning of full-length SgHMGR cDNA and its prokaryotic expression, and the recombined prokaryotic expression vector of SgHMGR was constructed successfully and could be expressed in BL21(DE3)of E.coli, which would lay foundation for further understanding of SgHMGR gene function and molecular regulation in mogroside V biosynthesis and provide reference about HMGR gene function study for other non-model plants.