CAJ | 학술논문

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1926 bp 的全长序列,经过生物信息学软件分析,发现该基因含有1749 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 Sg HMGR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 Sg HMGR 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。
라한과첨감 V 시일충호호완형사배삼첩류물질,작위주요적활성성분화첨미성분존재우성숙과실중,3-간기-3-갑기무이산단선보매 A 환원매(HMGR )작위첩류화합물생물합성도경중적제일개한속매,위우갑간무산(MVA)도경중,시라한과첨감 V 생물합성도경중적중요조공위점。위료심입료해라한과첨감 V 적생물합성도경,해연구종라한과전록조수거중획득일조편마 HMGR 적 unigene,이수분후3 d 적유과작위실험재료,통과 RACE 기술획득료1926 bp 적전장서렬,경과생물신식학연건분석,발현해기인함유1749 bp적개방열독광,편마582안기산잔기,함2단과막구,분별위우50~72 aa 화93~115 aa,아세포정위예측위우질막혹내질망상,예측해단백몰유신호태,계통진화수분석현시여동과식물황과화첨과중 HMGR 기인적동원성최고。해연구채취거도 N 단과막구적방법,구건원핵표체재체전화대장간균 BL21(DE3),경 IPTG 유도재상청화침정중균유융합단백출현,우기재25℃유도과야후상청중표체최명현。해문시수차대 Sg HMGR기인전장서렬적극륭급원핵표체적공능험증,위진일보심화 Sg HMGR 기인재라한과첨감 V 생물합성도경중적공능급분자조공연구타하기출。
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 ex-ists in the ripe fruits of S .grosvenorii .In the proposed mogoroside V biosynthetic pathway,there are two independ-ent pathways for triterpenoid biosynthesis (MVA pathway and MEP pathway).3-Hydroxy-3-methylglutaryl coen-zyme-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 bi-osynthetic 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 endo-plasm.By HMGR gene structure prediction analysis,the predicted SgHMGR protein had two transmembrane do-mains in N-terminal which were located in 50-72 aa and 93-115 aa,respectively.Besides,there were no predicted sig-nal peptides for SgHMGR protein.In order to avoid the influence of transmembrane domains on the heterologous ex-pression,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 success-fully and could be expressed in BL21(DE3)of E .coli ,which would lay foundation for further understanding of SgH-MGR gene function and molecular regulation in mogroside V biosynthesis and provide reference about HMGR gene function study for other non-model plants.