胶孢炭疽菌ES026石杉碱甲合成途径关键酶基因CgCAO、CgLDC的克隆和功能验证

发布时间：2018-04-03 06:28

本文选题：生物合成　切入点：赖氨酸脱羧酶　出处：《华中农业大学》2017年博士论文

【摘要】：石杉碱甲(Huperzine A)作为新一代治疗阿尔茨海默病的药物具有较好的前景,最先是从石松科植物中发现并提取,但由于人工合成石杉碱甲技术不成熟,植物资源匮乏,所以其产量受到了极大的限制。近些年来,很多学者分离报道很多产石杉碱甲的内生真菌,由于这一报道结果,因此分离产石杉碱甲的内生菌已成为新的热点。本实验材料是根据在前期研究试验中从蛇足石杉植物中分离得到了一株产石杉碱甲的内生真菌胶孢炭疽菌ES026,并已经申请专利。在本研究中,根据前期对胶孢炭疽菌ES026的基因组数据和转录组测序数据分析及基因功能注释结果,得出赖氨酸脱羧酶和铜胺氧化酶被注释到石杉碱甲生物合成路径中,为了验证赖氨酸脱羧酶和铜胺氧化酶的功能,本研究主要做了一下几个方面的内容:1.成功在胶孢炭疽菌ES026中克隆了赖氨酸脱羧酶基因(CgLDC)和铜胺氧化酶基因(CgCAO),CgLDC序列全长769 bp,CgCAO序列全长2072 bp。2.CgLDC和CgCAO的体外异源表达与蛋白纯化构建异源表达载体:pET28a-CgLDC和pET28a-CgCAO,转化至大肠杆菌表达菌株BL21(DE3)中。优化了蛋白表达条件,并利用优化后的蛋白纯化方法得到纯化后的CgLDC蛋白和CgCAO蛋白进行体外酶促活性反应,反应产物通过LC-MS分析,发现CgLDC具有催化L-Lysine生成尸胺的能力,CgCAO具有催化尸胺生成△1-Piperideine的能力。3.胶孢炭疽菌ES026中CgLDC基因和CgCAO基因体内过表达采用根癌农杆菌介导的转化方法将含有不同启动子的重组质粒转化到胶孢炭疽菌ES026中过表达CgLDC基因和CgCAO基因,经过抗性筛选和PCR验证得到10株突变株,利用实时定量PCR检测不同突变菌株CgLDC和CgCAO基因的表达量,通过LC-MS检测基因表达量与石杉碱甲产量的关系。
[Abstract]:Huperzine A (Huperzine A) is a promising drug for the treatment of Alzheimer's disease. Huperzine A was first found and extracted from Pinaceae plants, but due to the immaturity of synthetic Huperzine A technology, the plant resources are scarce.So its output is greatly restricted.In recent years, many scholars have isolated and reported many endophytic fungi producing Huperzine A. As a result of this report, the isolation of endophytic fungi from Huperzine A has become a new hotspot.In this study, an endophytic fungus ES026, an endophytic fungus producing Huperzine A, was isolated from Cunninghamia serrata in previous studies and has been patented.In this study, based on the analysis of genomic data and transcriptional sequence data of ES026 and the results of gene function annotation, it was concluded that lysine decarboxylase and copper amine oxidase were annotated into the biosynthesis pathway of Huperzine A.In order to verify the function of lysine decarboxylase and copper amine oxidase, this study mainly studied several aspects: 1.鎴愬姛鍦ㄨ兌瀛㈢偔鐤借弻ES026涓厠闅嗕簡璧栨皑閰歌劚缇ч叾鍩哄洜(CgLDC)鍜岄摐鑳烘哀鍖栭叾鍩哄洜(CgCAO),CgLDC搴忓垪鍏ㄩ暱769 bp,CgCAO搴忓垪鍏ㄩ暱2072 bp.2.CgLDC鍜孋gCAO鐨勪綋澶栧紓婧愯〃杈句笌铔嬬櫧绾寲鏋勫缓寮傛簮琛ㄨ揪杞戒綋:pET28a-CgLDC鍜宲ET28a-CgCAO,杞寲E. coli expression strain BL21 (DE3).The protein expression conditions were optimized, and the purified CgLDC protein and CgCAO protein were obtained by using the optimized protein purification method. The reaction products were analyzed by LC-MS.It was found that CgLDC has the ability to catalyze the formation of cadaverine by L-Lysine. CgCAO has the ability of catalyzing the formation of 1-Piperideine by CgCAO.Overexpression of CgLDC gene and CgCAO gene in ES026 of Bacillus anthracis was carried out by Agrobacterium tumefaciens mediated transformation. The recombinant plasmids containing different promoters were transformed into CgLDC gene and CgCAO gene in ES026.Ten mutants were obtained by resistance screening and PCR validation. The expression of CgLDC and CgCAO genes in different mutant strains was detected by real-time quantitative PCR. The relationship between the expression of CgLDC and CgCAO gene and the yield of Huperzine A was detected by LC-MS.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q936

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