基于喜树碱生物合成的喜树内生菌及其关键酶基因克隆
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摘要
以喜树内生菌为研究对象,经发酵培养后提取喜树碱及其衍生物,采用HPLC法对发酵液中喜树碱及其衍生物进行定性检测,获得能够产生喜树碱(CPT)及10-羟基喜树碱(HCPT)的喜树内生菌。通过对喜树碱生物合成途径的研究,选取10-羟基香叶醇羟化酶(G10H)、10-羟基香叶醇脱氢酶(HGO)、色氨酸脱羧酶(TDC)和异胡豆苷合酶(STR)4个关键酶基因作为研究对象,设计特异性引物,克隆目的基因。提取喜树RNA,经反转录合成cDNA,同时提取喜树内生菌DNA。分别以植物cDNA和内生菌DNA作为模板,筛选鉴定喜树碱合成途径的关键酶基因G10 H、HGO、TDC和STR并与喜树关键酶基因进行比较,初步解析喜树碱代谢途径,为喜树内生菌发酵合成喜树碱及其衍生物提供理论依据。
Camptothecin and its derivatives were extracted from the endophytic bacteria from Camptotheca acuminata,and qualitative determined by HPLC.The results showed that two endophytic bacteria of C.acuminata could produce camptothecin and 10-hydroxy camptothecin.Four key enzyme genes,including G10 H,HGO,TDC,STR,were selected as the targets.The specific primers were designed to clone the target gene in order to confirm the synthetic pathway of camptothecin.The RNA of C.acuminata and the DNA of endophytic bacterium fromC.acuminata were extracted.The cDNA of C.acuminata was synthesized by reverse transcription method.Four key enzymes genes,G10 H,HGO,TDCand STR,in camptothecin metabolic pathway,were cloned and compared with those of C.acuminata using plant cDNA and endophytic DNA as templates.The metabolic pathway of camptothecin was preliminary analyzed.The results provided a theoretical foundation for synthesis of camptothecin and its derivatives by endophytes.
Camptothecin and its derivatives were extracted from the endophytic bacteria from Camptotheca acuminata,and qualitative determined by HPLC.The results showed that two endophytic bacteria of C.acuminata could produce camptothecin and 10-hydroxy camptothecin.Four key enzyme genes,including G10 H,HGO,TDC,STR,were selected as the targets.The specific primers were designed to clone the target gene in order to confirm the synthetic pathway of camptothecin.The RNA of C.acuminata and the DNA of endophytic bacterium fromC.acuminata were extracted.The cDNA of C.acuminata was synthesized by reverse transcription method.Four key enzymes genes,G10 H,HGO,TDCand STR,in camptothecin metabolic pathway,were cloned and compared with those of C.acuminata using plant cDNA and endophytic DNA as templates.The metabolic pathway of camptothecin was preliminary analyzed.The results provided a theoretical foundation for synthesis of camptothecin and its derivatives by endophytes.
引文
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[8]杨帆,李鹤,杨兰,等.一种新型α-甘露糖苷酶的异源表达及酶学性质表征[J].大连工业大学学报,2018,37(5):320-325.
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[10]王磊,龙秀峰,肖青,等.一株10-羟基喜树碱转化内生菌的筛选及鉴定[J].生物技术通报,2014,24(1):168-176.
[11]VENUGOPALAN A,SRIVASTAVA S.Enhanced camptothecin production by ethanol addition in the suspension culture of the endophyte,Fusarium solani[J].Bioresource Technology,2015,188(2):251-257.
[12]KUSARI S,ZVHLKE S,SPITELLER M.Effect of artificial reconstitution of the interaction between the plant Camptotheca acuminata and the fungal endophyte Fusarium solani on camptothecin biosynthesis[J].Journal of Natural Products,2011,74(4):764-75.
[13]ROTH J,LEROITH D,COLLIER E S,et al.The evolutionary origins of intercellular communication and the maginot lines of the mind[J].Annals of the New York Academy of Sciences,1986,463(2):1-11.
[2]陈道金,刘慧敏,杨静,等.植物内生菌活性成分研究进展[J].时珍国医国药,2013,24(7):23-29.
[3]王磊,吴家胜,廖亮.喜树碱生物合成途径及其相关酶研究现状及展望[J].浙江林学院学报,2008,25(6):791-797.
[4]STROBEL G A.Endophytes as sources of bioactive products[J].Microbes and Infection,2003,5(6):535-544.
[5]AMBROSE C,VARGHESE C,SUBHASH J B,et al.Endophytic bacteria as a source of novel antibiotics:an overview[J].Pharmacognosy Reviews,2013,7(13):11-16.
[6]WALL M E,WANI M C,COOK C E,et al.Plant antitumor agents,the isolation and structure of camptothecin,a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata[J].Journal of the American Chemical Society,1966,88(16):3888-3890.
[7]李连强,潘夕春,谈锋.喜树碱的生物合成途径及生物技术研究进展[J].中草药,2006,37(4):623-626.
[8]杨帆,李鹤,杨兰,等.一种新型α-甘露糖苷酶的异源表达及酶学性质表征[J].大连工业大学学报,2018,37(5):320-325.
[9]赵庆云,樊明涛,师俊玲.产黄酮银杏内生菌的分离与鉴定[J].西北农业学报,2007,16(2):169-173.
[10]王磊,龙秀峰,肖青,等.一株10-羟基喜树碱转化内生菌的筛选及鉴定[J].生物技术通报,2014,24(1):168-176.
[11]VENUGOPALAN A,SRIVASTAVA S.Enhanced camptothecin production by ethanol addition in the suspension culture of the endophyte,Fusarium solani[J].Bioresource Technology,2015,188(2):251-257.
[12]KUSARI S,ZVHLKE S,SPITELLER M.Effect of artificial reconstitution of the interaction between the plant Camptotheca acuminata and the fungal endophyte Fusarium solani on camptothecin biosynthesis[J].Journal of Natural Products,2011,74(4):764-75.
[13]ROTH J,LEROITH D,COLLIER E S,et al.The evolutionary origins of intercellular communication and the maginot lines of the mind[J].Annals of the New York Academy of Sciences,1986,463(2):1-11.