蛇足石杉内生真菌Shiraia sp.Slf14中LDC和CAO基因的克隆及表达

发布时间：2018-03-18 21:10

本文选题：石杉碱甲　切入点：内生真菌　出处：《江西师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：石杉碱甲(Huperzine A,HupA)是治疗阿尔茨海默症的生物碱类药物。由于化学合成、石松属植物栽培及细胞培养等生产技术无法突破,HupA仅能从野生蛇足石杉等石松属植物中提取,存在原料短缺和环境破坏等严重问题,内生真菌发酵生产HupA成为可供选择的替代方式。生物合成途径的阐明是采用基因组学、代谢工程、组合生物合成等现代生物技术生产代谢产物的基础,但HupA等石松生物碱生物合成途径尚未阐明。本研究以高产HupA的蛇足石杉内生真菌Shiraia sp.Slf14为对象,对全基因组测序和生物信息学分析预测的编码石杉碱甲生物合成中第一、二步反应的关键酶—赖氨酸脱羧酶(Lysine decarboxylase,LDC)和铜胺氧化酶(copper amine oxidase,CAO)基因,开展其体外功能分析。根据全基因组序列信息获得的LDC序列信息,本研究采用RT-PCR技术,从Shiraia sp.Slf14中扩增得到长度为687 bp的LDC基因(GenBank ID:KT362170),并成功构建大肠杆菌表达质粒pET-22b-LDC与pET-32a-LDC,转化感受态细胞E.coli BL21(DE3),诱导表达出重组蛋白LDC与Trx-LDC。经SDS-PAGE电泳鉴定分子量分别约24 kDa和42 kDa,与预计大小相符。通过Ni2+金属亲和层析纯化重组LDC与Trx-LDC并建立酶促反应体系,利用TLC检测LDC催化活性,结果表明LDC与Trx-LDC均具有赖氨酸脱羧酶活性。利用生物信息学软件分析了LDC的理化性质及蛋白质的空间结构:二级结构显示出α螺旋占37.3%,β折叠占11.4%,无规则卷曲为51.3%;从三维结构看出LDC为二聚体结构,N端肽链暴露在蛋白表面,主要通过C端肽链相互作用形成二聚体。基于赖氨酸脱羧酶的氨基酸序列系统进化树分析可知,Shiraia sp.Slf14的LDC与来源于Aspergillus clavatus NRRL 1的相应蛋白的氨基酸序列相似度最高,和来自宿主植物Huperzia serrata的两个LDC差异较大。基于全基因组序列信息获得的CAO基因相关信息,采用RT-PCR技术扩增得到铜胺氧化酶(SsCAO)基因,基因长度2031 bp,序列已提交至NCBI数据库(GenBank ID:KT362171)。成功构建大肠杆菌表达载体pET-SsCAO并转化感受态细胞E.coli BL21(DE3),经诱导表达出76 kDa大小的重组SsCAO蛋白质,与预测大小相吻合。重组SsCAO经纯化后构建酶促反应体系,GC-MS检测发现了产物5-氨基戊醛(5-aminopentanal),表明重组SsCAO具有催化活性。通过生物信息学分析其理化性质和预测蛋白质的结构,SsCAO蛋白质二级结构和三维结构显示α-螺旋较集中于C/N两端,β-折叠结构所占比例较大。分析SsCAO的氨基酸序列发现,其含有CAO家族的保守序列Asn-Tyr-Asp/Glu,His447、His449、His607和Tyr299,Lys309 and Asp313。系统发育树分析表明SsCAO与匐柄霉属Stemphylium lycopersici CAO关系最近,与H.serrata CAO和其他的植物CAOs相距较远。本文对LDC和SsCAO的研究不仅为内生真菌石杉碱甲生物合成机制提供参考数据,同时也为HupA的合成生物学研究奠定基础。
[Abstract]:Huperzine (Huperzine HupA) is an alkaloid drug for the treatment of Alzheimer's disease. Due to chemical synthesis, the production techniques such as cultivation and cell culture of Pinus can not break through the production of HupA. There are serious problems such as shortage of raw materials and environmental damage. Endophytic fungi fermentation to produce HupA is an alternative alternative. The biosynthesis pathway is elucidated by using genomics, metabolic engineering, and so on. The basis of production of metabolites by combining biosynthesis and other modern biological techniques, but the biosynthesis pathway of alkaloids of Pinus lanceolata such as HupA has not been elucidated. In this study, the endophytic fungus Shiraia sp.Slf14, which produces high HupA, was used as an object. The Lysine decarboxylase LDCand copper amine oxidase copper amine oxidase CAO genes were predicted for the first and second step reaction of Huperzine A biosynthesis by whole genome sequencing and bioinformatics analysis. According to the LDC sequence information obtained from the whole genome sequence information, the RT-PCR technique was used in this study. A 687bp LDC gene named IDKT362170 was amplified from Shiraia sp.Slf14. The E. coli expression plasmids pET-22b-LDC and pET-32a-LDCwere successfully constructed, and the recombinant protein LDC and Trx-LDCwere induced to express the recombinant protein LDC and Trx-LDC. the molecular weights were about 24 kDa by SDS-PAGE electrophoresis. The recombinant LDC and Trx-LDC were purified by Ni2 metal affinity chromatography and the enzymatic reaction system was established. TLC was used to detect the catalytic activity of LDC. The results showed that both LDC and Trx-LDC had lysine decarboxylase activities. The physicochemical properties and protein spatial structure of LDC were analyzed by bioinformatics software. The secondary structure showed that 伪 helix was 37.3cm, 尾 folding 11.4g and irregular crimp 51.3; According to the three-dimensional structure, the N-terminal peptide chain of LDC is a dimer structure exposed to the surface of the protein. The amino acid sequence phylogenetic tree analysis based on lysine decarboxylase showed that LDC of Shiraia sp.Slf14 had the highest amino acid sequence similarity with the corresponding protein from Aspergillus clavatus NRRL 1. Based on the relative information of CAO gene obtained from the whole genome sequence, the copper amine oxidase gene SsCAO) gene was amplified by RT-PCR technique. The length of the gene was 2031 BP, and the sequence was submitted to the NCBI database (GenBank: IDKT362171). The E. coli expression vector pET-SsCAO was successfully constructed and transformed into E. coli BL21DE-3, and the recombinant SsCAO protein with the size of 76 kDa was induced and expressed. After purification of recombinant SsCAO, the enzymatic reaction system (GC-MS) was used to detect the product of 5-aminopentanalanalen, which indicated that the recombinant SsCAO had catalytic activity. The physicochemical properties of recombinant SsCAO were analyzed by bioinformatics and the predicted protein was predicted. The secondary and three-dimensional structures of SsCAO protein showed that 伪 -helix was concentrated at the two ends of C / N, and 尾 -fold structure occupied a large proportion. The amino acid sequence of SsCAO was found. The conserved sequences of Asn-Tyr-Asp.Gluus His447, His449, His607 and Tyr299Lys309 and Asp313. phylogenetic tree analysis showed that SsCAO had the closest relationship with Stemphylium lycopersici CAO. The study on LDC and SsCAO not only provided reference data for the biosynthesis mechanism of endophytic fungi Huperzine A, but also laid a foundation for the study of synthetic biology of HupA.
【学位授予单位】：江西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q93;Q78

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