本文選題：生物合成　切入點(diǎn)：賴氨酸脫羧酶　出處：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：石杉?jí)A甲(Huperzine A)作為新一代治療阿爾茨海默病的藥物具有較好的前景,最先是從石松科植物中發(fā)現(xiàn)并提取,但由于人工合成石杉?jí)A甲技術(shù)不成熟,植物資源匱乏,所以其產(chǎn)量受到了極大的限制。近些年來,很多學(xué)者分離報(bào)道很多產(chǎn)石杉?jí)A甲的內(nèi)生真菌,由于這一報(bào)道結(jié)果,因此分離產(chǎn)石杉?jí)A甲的內(nèi)生菌已成為新的熱點(diǎn)。本實(shí)驗(yàn)材料是根據(jù)在前期研究試驗(yàn)中從蛇足石杉植物中分離得到了一株產(chǎn)石杉?jí)A甲的內(nèi)生真菌膠孢炭疽菌ES026,并已經(jīng)申請(qǐng)專利。在本研究中,根據(jù)前期對(duì)膠孢炭疽菌ES026的基因組數(shù)據(jù)和轉(zhuǎn)錄組測(cè)序數(shù)據(jù)分析及基因功能注釋結(jié)果,得出賴氨酸脫羧酶和銅胺氧化酶被注釋到石杉?jí)A甲生物合成路徑中,為了驗(yàn)證賴氨酸脫羧酶和銅胺氧化酶的功能,本研究主要做了一下幾個(gè)方面的內(nèi)容:1.成功在膠孢炭疽菌ES026中克隆了賴氨酸脫羧酶基因(CgLDC)和銅胺氧化酶基因(CgCAO),CgLDC序列全長(zhǎng)769 bp,CgCAO序列全長(zhǎng)2072 bp。2.CgLDC和CgCAO的體外異源表達(dá)與蛋白純化構(gòu)建異源表達(dá)載體:pET28a-CgLDC和pET28a-CgCAO,轉(zhuǎn)化至大腸桿菌表達(dá)菌株BL21(DE3)中。優(yōu)化了蛋白表達(dá)條件,并利用優(yōu)化后的蛋白純化方法得到純化后的CgLDC蛋白和CgCAO蛋白進(jìn)行體外酶促活性反應(yīng),反應(yīng)產(chǎn)物通過LC-MS分析,發(fā)現(xiàn)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.膠孢炭疽菌ES026中CgLDC基因和CgCAO基因體內(nèi)過表達(dá)采用根癌農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化方法將含有不同啟動(dòng)子的重組質(zhì)粒轉(zhuǎn)化到膠孢炭疽菌ES026中過表達(dá)CgLDC基因和CgCAO基因,經(jīng)過抗性篩選和PCR驗(yàn)證得到10株突變株,利用實(shí)時(shí)定量PCR檢測(cè)不同突變菌株CgLDC和CgCAO基因的表達(dá)量,通過LC-MS檢測(cè)基因表達(dá)量與石杉?jí)A甲產(chǎn)量的關(guān)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(jiǎn)璧栨皚閰歌劚緹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〃杈句笌铔嬬櫧綰寲鏋勫緩寮傛簮琛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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q936

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