一株苝醌类光敏剂产生菌AL18的cDNA文库的构建
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摘要
本研究以一株采自云南西部山区，次生代谢产物为苝醌类衍生物光敏剂的丝状真菌AL18为实验材料，在确立了其总RNA提取方法的基础上，构建了cDNA文库，主要结果如下：
1．为克服RNA易降解，丝状真菌富含RNase(RNA酶)和多糖的困难，在参考不同实验材料的总RNA提取方法的基础上，通过实验确立了采用异硫氰酸胍和β-巯基乙醇联合变性，酚、氯仿、异戊醇多次抽提，使RNA留在水相而DNA和蛋白质进入有机相，然后用异丙醇沉淀RNA的方法，得到了完整、均一的总RNA，质量能够满足构建cDNA文库的要求。
2．利用MMLV反转录酶把总RNA中的mRNA反转录成cDNA，用特异引物进行LD-PCR扩增合成双链cDNA；用CHROMA SPIN-400Column对cDNA进行分级分离，去除小于500bp的cDNA片段；用带有粘性末端的双链cDNA与λTriplEx2载体连接，利用λ噬菌体包装蛋白对合成的双链cDNA进行体外包装，包装产物侵染E．coli XL-Blue获得cDNA文库。该cDNA文库的滴度为1.04×10~6pfu／ml，扩增后文库的滴度为1.32×10~(10)pfu／ml，蓝白斑筛选证实其重组率为82％。同时，由于反转录过程中采用附加序列的方法使全长mRNA得到反转录，所以得到的cDNA是完整的，有利于各种基因的全长cDNA的获得。为筛选文库克隆苝醌类光敏剂生物合成中的聚酮合成酶基因以及在后续工作中克隆聚酮合成酶的全基因簇打下坚实的基础。
This work uses a fungus AL18 producing Perylenequinonoid derivatives photosensitizer which was picked from the mountainous area at the western part of Yunnan.On the basis of establishing the method of total RNA extraction, the cDNA library is constructed.The main results are as follows:
1. In order to get over the problem that RNA is easy to degrade and there are a lot of RNase and glucide in this fungus. On the basis of consulting many methods of total RNA extraction, the method is established by experiment for total RNA extraction using guanidinium isothiocyante and β-mercaptoethanol for denaturation, then using water-satured phenol ,chloroform and isoamylalcohole for extraction, RNA stay in water ,while DNA and protein stay in another medium. Finally, RNA is deposited by isopropylalcohol .The integrity and purification of the total RNA isolated are eligible and meet the requirement to construct cDNA library.
2. The cDNA is synthesized from the total RNA with the one point mutant of Moloney murine leukemia virus(MMLV) reverse transcriptase,and the cDNA is further size-fractioned by CHROMA SPIN-400 Column to remove the fraction shorter than 500bp. The dscDNA is cloned into the λTriplEx2 vector and the resulting ligating matarial is λ-phage packaged. The unamplifed cDNA library is titered using E.coli XL-Blue, and it turns out that the titer of the cDNA library is 1.04 ×10~6 pfu/ml,the titer of the amplified cDNA library isl.32×10~(10)pfu/ml,and the recombinant efficiency is 82% through white/blue screening.It is available to clone the polyketide biosynthase gene for Polyketide biosynthesis.
1．为克服RNA易降解，丝状真菌富含RNase(RNA酶)和多糖的困难，在参考不同实验材料的总RNA提取方法的基础上，通过实验确立了采用异硫氰酸胍和β-巯基乙醇联合变性，酚、氯仿、异戊醇多次抽提，使RNA留在水相而DNA和蛋白质进入有机相，然后用异丙醇沉淀RNA的方法，得到了完整、均一的总RNA，质量能够满足构建cDNA文库的要求。
2．利用MMLV反转录酶把总RNA中的mRNA反转录成cDNA，用特异引物进行LD-PCR扩增合成双链cDNA；用CHROMA SPIN-400Column对cDNA进行分级分离，去除小于500bp的cDNA片段；用带有粘性末端的双链cDNA与λTriplEx2载体连接，利用λ噬菌体包装蛋白对合成的双链cDNA进行体外包装，包装产物侵染E．coli XL-Blue获得cDNA文库。该cDNA文库的滴度为1.04×10~6pfu／ml，扩增后文库的滴度为1.32×10~(10)pfu／ml，蓝白斑筛选证实其重组率为82％。同时，由于反转录过程中采用附加序列的方法使全长mRNA得到反转录，所以得到的cDNA是完整的，有利于各种基因的全长cDNA的获得。为筛选文库克隆苝醌类光敏剂生物合成中的聚酮合成酶基因以及在后续工作中克隆聚酮合成酶的全基因簇打下坚实的基础。
This work uses a fungus AL18 producing Perylenequinonoid derivatives photosensitizer which was picked from the mountainous area at the western part of Yunnan.On the basis of establishing the method of total RNA extraction, the cDNA library is constructed.The main results are as follows:
1. In order to get over the problem that RNA is easy to degrade and there are a lot of RNase and glucide in this fungus. On the basis of consulting many methods of total RNA extraction, the method is established by experiment for total RNA extraction using guanidinium isothiocyante and β-mercaptoethanol for denaturation, then using water-satured phenol ,chloroform and isoamylalcohole for extraction, RNA stay in water ,while DNA and protein stay in another medium. Finally, RNA is deposited by isopropylalcohol .The integrity and purification of the total RNA isolated are eligible and meet the requirement to construct cDNA library.
2. The cDNA is synthesized from the total RNA with the one point mutant of Moloney murine leukemia virus(MMLV) reverse transcriptase,and the cDNA is further size-fractioned by CHROMA SPIN-400 Column to remove the fraction shorter than 500bp. The dscDNA is cloned into the λTriplEx2 vector and the resulting ligating matarial is λ-phage packaged. The unamplifed cDNA library is titered using E.coli XL-Blue, and it turns out that the titer of the cDNA library is 1.04 ×10~6 pfu/ml,the titer of the amplified cDNA library isl.32×10~(10)pfu/ml,and the recombinant efficiency is 82% through white/blue screening.It is available to clone the polyketide biosynthase gene for Polyketide biosynthesis.
引文
[1] 张红雨，赵红霞，张志义．用ESR方法研究光敏作用的动态过程．中国科学(C辑)．1997，27(5)：398～403．
[2] 蒋丽金，何玉英．竹红菌素类光敏剂的光物理、光化学及光生物．科学通报 2000，45(19)：2019～2033．
[3] 罗子华，于兰馥，郭泳华，等．竹红菌治疗外阴白病变的临床观察．云南医药．1990，1(1)：20～25．
[4] 于兰馥，罗子华．张胜泉，等．竹红菌软膏加光治疗外阴病变及其组织变化的研究．中华妇产科杂志，1984，19(1)：29～31．
[5] 王家壁，包建南．皮肤淀粉样变苔藤37例临床分析及竹红菌光化疗法治疗观察．中国医学科学院学报，1985，5(7)：349～351．
[6] 梁睿媛，梅国栋，杨正中，等．竹红菌光化疗治疗肥厚性瘢痕62例报告．中华皮肤科杂志，1982，15(2)：87～88．
[7] Hudson JB, Imperial V, Haugland RP, et al. Antiviral Activities of Photoactive Perylenequinones. Photochemistry and Photobiology. 1997, 65(2): 352～354.
[8] Miller G, Brown K, Ballangrud AM, et al. Preclinical Assessment Hypocrellin B and Hypocrellin B Derivatives as Sensitizers for Photodynamic Therapy of Cancer: progress Update. Photochemistry and Photobiology. 1997, 65(4): 714～722.
[9] Hirayama J, Kenji Ikebuchi Hil-Won kwon. Photo inactivation of Virus infectivity by Hypocrellin A. Photochemistry and Photobiology. 1997, 65(5): 697～700.
[10] Hudson JB, Zhov J, Chen J. Hypocrellin from Hypocrell A Bambuase is Phototoxic to Human Immunodeficiency Virus. Photochemistry and Photobiology. 1994, 60(3): 253～255.
[11] Li LM, Zhang ZY, Wang DH, et al. The photonduced electron transfer between hypollins and colloidal semiconductors 1. Kinetics of photosensitized reduction in a collidal CDS system with hypollina as a sensitizer. J Photochem Photobiol, A: Chem, 1997, 103: 279～284.
[12] 邱勇，宋心琦．光话化农药．化学通报．1993(1)：21～24．
[13] 王秀丽，孙振令．刘为忠，张红雨．四种苝醌类衍生物光敏活性的比较．山东理工大学学报(自然科学版)．2003．17(5)：21～24
[14] 卢明锋．一株苝醌类光敏剂产生菌的rRNA基因ITS测序分析及液体深层发酵工艺研．[学位论文]．四川雅安．四川农业大学．2004．
[15] 肖竦．肿瘤光动力疗法．实用癌症杂志．2005．20(2)：214～216．
[16] 程极济．光生物物理学．北京：高等教育出版社，1987：98．
[17] Cox GS, Whitten OG, kessel D, et al. Porphyrin photopsitizaion. New York: Plenum Press, 1983, 227～240.
[18] Dougherty TJ. An update on photodynamic therapy applications. J Clin Laser Med Sury, 2002, 20(1): 3.
[19] Hayata YH, Kato. Overview of clinical PDT. In Photodynamic therapy and biomedical lasers. Amsterdam: Eleevier Science Publishers, 1992: 1～5.
[20] Jula G, Levy AL. New Applications in Photodynamic therapy introduction. Photochemistry and Photobiology, 1996, 64(5): 737.
[21] Barber P, Barr H, George J, et al. Photodynamic therapy in the treatment of lung and oesophageal cancers. Clin Oncol (R CollRadiol), 2002, 14(2): 110.
[22] 翟力平，李世荣．激光诱发荧光光谱结对肠癌的诊断．中华肿瘤杂志，1998，20(1)：76．
[23] 阮珊真，盛月霞．光敏疗法治疗颈淋巴结转移癌2例报道．肿瘤，1989，9(5)：216．
[24] 陈煜清，李长岭，刘永志，等．抗坏血酸对血卟啉衍生物渗入肿瘤组织及其光敏作用的影响．中华肿瘤杂志，1997，19(5)：350．
[25] Giulio J. Tumout photo sensitizers: approaches to enhance the selectivity and efficiency of photodynamic therapy. J Photochem Photobiol B: Biol, 1996, 36: 87.
[26] Bierbaum G, Fuchs K, Lenz W, et al. Presence of Staphylococcus aureus with reduced susceptibility to vancomycin in Germany. Eur JC lin Microbiol InfectD is, 1999, 18: 691.
[27] Hassan IA, Chadwick PR, Johnson A P. Clinical isolates of methicillin resistant Staphylococcus aureus (MRSA)with reduced susceptibility to teicoplanin in Northwest England. J A ntim icrob Chem other, 2001, 48: 454.
[28] Hiramatsu K. The emergence of Staphylococcus aureus with reduced susceptibility to vancomycin in Japan. Am J Med, 1998, 104: 78.
[29] Zeina B, Greenman J, Purcell WM, et al. Killing of cutaneousm icrobial species by photodynamic therapy. B r J Dermatol, 2002, 146(4):568.
[30] 张红雨，刘为忠．北醌衍生物光敏生物杀菌剂及其制法．中国专利．申请号：021110386．0，2001．
[31] Yoshimura M, Namura S, Akamatsu H, et al. Antimicrobial effects of photo therapy and photochemo therapy in vivo and in vitro. B r J Dermatol, 135:528.
[32] Gasparro FP. Psoralen photobiology: recent advances. Photochem Photobiol, 1996, 63: 553.
[33] Fehr MJ, McCoskey MA, Petrich JW. Light induce dacidification by antiviral agent hypericin. J Am Chem Soc, 1995, 117: 1833.
[34] Lavie G, Mazur Y, Lavie D, et al. Hypericin as an inactivator of infectious viruses in blood components. Transfusion, 1995, 35(5): 392.
[35] North J, Neyndorff H, Levy JG. Photo sensitizers as virucidal agents. J Photochem Photobiol B:Biol, 1993, 17:99.
[36] Nitzan Y, Balzam R, Sudakevitz A, Ashkenazi H. Eradication of Acine to batter baumannii by photo sensitized agents in vitro. J Photochem Photobiol B: Biol, 1998, 42:211.
[37] Saeki Y, Ito Y, Shibata M, et al. Antimicrobial action of green tea extract, flavono flavor and copperchlo rophyll against oral bacteria. Bull Tokyo Dent Coll, 1993, 34(1):33.
[38] 唐蕾，诸葛建．天然光敏剂的光动力抗菌作用．中国抗生素杂志．2004，29(11)：703～706．
[39] 何玉英．科学通报．竹红菌素衍生物在缺氧条件下的光化学．1999，44(22)：2376～2383．
[40] 蒋丽金．竹红菌素的结构、性质、光化学反应及反应机制．科学通报，1990，35(21)：1608～1616．
[41] Chao C, Zhang P. A Facile Route to Peylenquinone. Tetra Lett. 1988, 29(2):225.
[42] Zhao C, Zhang XS, Zhang P. Some Synthetic Studies Related to Peryenequinones. Liebigs Ann Chemt, 1993, 35～41.
[43] 付滨，张傍．天然苝醌合成的初步研究，北京大学学报，2000，36(2)：167-171．
[44] 第五震军，蒋丽金，张曼华．竹红菌甲素光敏氧化反应机制．化学学报，1990，48：483-488．
[45] 朱晓微．贯叶金丝桃研究进展Ⅱ一药代动力学、药效学和临床应用(续)．国外医约·植物药分册，1998，13(5)：210～214．
[46] 李聪，陈远腾，林南英，等．一株产苝醌光敏化合物的真菌成分分析．菌物系统， 2000，19(1)，122～127．
[47] 刘为忠，李聪，谢金伦，等．苝醌类化合物产生菌固态发酵工艺的研究．云南大学学报(自科版)，2000，22(5)：389～391．
[48] Weber T, W elzel K, Pelzer S, et al. Exploiting the genetic potential of polyketide producing strep tomycetes. J Bio technol, 2003, 106 (2-3): 221
[49] 邵荣光，王以光．微生物组合生物合成药物的研究进展与趋势．天津药学．2004，16．(6) 1～3
[50] Ben S. Polyketide biosynthesis beyond the type Ⅰ, Ⅱ and Ⅲ polyketide synthase paradigms. Biocatalysis and biotransformation. 2003, 7: 285～295.
[51] Hopwood DA, Sherman DH. Molecular Genetics of polyketides and its comparison to Fatty acid biosynthesis. Annu Rev Genet, 1990, 24:37～66.
[52] 刘文，唐功利．以生物合成为基础的代谢工程和组合生物合成．中国生物工程杂志．2005(25) 1：1～5
[53] 黄曦，邓子新．聚酮分子生物学进展．国外医约抗生素分册．1998，19(3)：169～176．
[54] Cortes J, Haydock SF, Roberts GA, et al. An unusually large multifunctional polyketide in the erythrornycin—polyketide synthase of Saccharo Polyspora erythraea. Nature. 1990, 346: 176～178.
[55] Donadio S, Stayer MJ, McAlpine JB, Swanson SJ, Katz L. Modular organization of genes required for complex polyketide biosynthesis. Science, 1991, 252: 675～679.
[56] 汪星明，赵凤生．组合生物合成．国外医药抗生素分册．2003，24(2)：49～53．
[57] Nicholson TP, Rudd BA, Dawson M, et al. Design and utility of oligonucleotide gene probes for fungal polyketide synthases. Chem Biol. 2001,8: 157.
[58] Hopwood DA. Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico. Microbiology, 1999, 145:2183.
[59] John K, Borchard T. Genetic engineering may keep one of the richest drug gold mines from being played out. Modern Drag Discovery, 1999, 2:22.
[60] Sambrook J. Molecular Cloning: A laboratory manual, 2nd eds. Cold Spring Harbor Lab Press, 1989, 396～449.
[61] 汤祖辉，赖卫华，许杨．红曲霉RNA的提取方法．江西科学，2002，20(2)：118～120．
[62] 萨姆布鲁克J，弗里奇EF，曼尼阿蒂斯T．分子克隆实验指南(第二版)．北京：科学出版社，1999．343～367．
[63] 李明春，王俊琦，邢来君．丝状真菌深黄被孢霉RNA的提取方法．菌物系统，1999，18(1)：108～111．
[64] 张璘，鱼红闪，金凤燮．真菌中RNA的提取方法．大连轻工业学院学报，2004，23(1)：22～24
[65] 李想，杨奇志，张飞雄．一种小量提取植物总RNA的有效方法．生物技术通报，2004，2：49～51．
[66] Piero C, Catrine K, Akiko K, et al. High-efficiency full-length cDNA cloning by biotinylated CAP trapper, Genomics, 1996, 37:327～336
[67] 朱玉贤、李毅主编．现代分子生物学，北京：高等教育出版社，2002，7：185～189．
[68] Thomas P, Nicholson, Brian AM, et al. Design and utility of oligonucleotide gene probes for fungal polyketide synthases. Chemistry and Biology. 2001, 8(2):157～178.
[2] 蒋丽金，何玉英．竹红菌素类光敏剂的光物理、光化学及光生物．科学通报 2000，45(19)：2019～2033．
[3] 罗子华，于兰馥，郭泳华，等．竹红菌治疗外阴白病变的临床观察．云南医药．1990，1(1)：20～25．
[4] 于兰馥，罗子华．张胜泉，等．竹红菌软膏加光治疗外阴病变及其组织变化的研究．中华妇产科杂志，1984，19(1)：29～31．
[5] 王家壁，包建南．皮肤淀粉样变苔藤37例临床分析及竹红菌光化疗法治疗观察．中国医学科学院学报，1985，5(7)：349～351．
[6] 梁睿媛，梅国栋，杨正中，等．竹红菌光化疗治疗肥厚性瘢痕62例报告．中华皮肤科杂志，1982，15(2)：87～88．
[7] Hudson JB, Imperial V, Haugland RP, et al. Antiviral Activities of Photoactive Perylenequinones. Photochemistry and Photobiology. 1997, 65(2): 352～354.
[8] Miller G, Brown K, Ballangrud AM, et al. Preclinical Assessment Hypocrellin B and Hypocrellin B Derivatives as Sensitizers for Photodynamic Therapy of Cancer: progress Update. Photochemistry and Photobiology. 1997, 65(4): 714～722.
[9] Hirayama J, Kenji Ikebuchi Hil-Won kwon. Photo inactivation of Virus infectivity by Hypocrellin A. Photochemistry and Photobiology. 1997, 65(5): 697～700.
[10] Hudson JB, Zhov J, Chen J. Hypocrellin from Hypocrell A Bambuase is Phototoxic to Human Immunodeficiency Virus. Photochemistry and Photobiology. 1994, 60(3): 253～255.
[11] Li LM, Zhang ZY, Wang DH, et al. The photonduced electron transfer between hypollins and colloidal semiconductors 1. Kinetics of photosensitized reduction in a collidal CDS system with hypollina as a sensitizer. J Photochem Photobiol, A: Chem, 1997, 103: 279～284.
[12] 邱勇，宋心琦．光话化农药．化学通报．1993(1)：21～24．
[13] 王秀丽，孙振令．刘为忠，张红雨．四种苝醌类衍生物光敏活性的比较．山东理工大学学报(自然科学版)．2003．17(5)：21～24
[14] 卢明锋．一株苝醌类光敏剂产生菌的rRNA基因ITS测序分析及液体深层发酵工艺研．[学位论文]．四川雅安．四川农业大学．2004．
[15] 肖竦．肿瘤光动力疗法．实用癌症杂志．2005．20(2)：214～216．
[16] 程极济．光生物物理学．北京：高等教育出版社，1987：98．
[17] Cox GS, Whitten OG, kessel D, et al. Porphyrin photopsitizaion. New York: Plenum Press, 1983, 227～240.
[18] Dougherty TJ. An update on photodynamic therapy applications. J Clin Laser Med Sury, 2002, 20(1): 3.
[19] Hayata YH, Kato. Overview of clinical PDT. In Photodynamic therapy and biomedical lasers. Amsterdam: Eleevier Science Publishers, 1992: 1～5.
[20] Jula G, Levy AL. New Applications in Photodynamic therapy introduction. Photochemistry and Photobiology, 1996, 64(5): 737.
[21] Barber P, Barr H, George J, et al. Photodynamic therapy in the treatment of lung and oesophageal cancers. Clin Oncol (R CollRadiol), 2002, 14(2): 110.
[22] 翟力平，李世荣．激光诱发荧光光谱结对肠癌的诊断．中华肿瘤杂志，1998，20(1)：76．
[23] 阮珊真，盛月霞．光敏疗法治疗颈淋巴结转移癌2例报道．肿瘤，1989，9(5)：216．
[24] 陈煜清，李长岭，刘永志，等．抗坏血酸对血卟啉衍生物渗入肿瘤组织及其光敏作用的影响．中华肿瘤杂志，1997，19(5)：350．
[25] Giulio J. Tumout photo sensitizers: approaches to enhance the selectivity and efficiency of photodynamic therapy. J Photochem Photobiol B: Biol, 1996, 36: 87.
[26] Bierbaum G, Fuchs K, Lenz W, et al. Presence of Staphylococcus aureus with reduced susceptibility to vancomycin in Germany. Eur JC lin Microbiol InfectD is, 1999, 18: 691.
[27] Hassan IA, Chadwick PR, Johnson A P. Clinical isolates of methicillin resistant Staphylococcus aureus (MRSA)with reduced susceptibility to teicoplanin in Northwest England. J A ntim icrob Chem other, 2001, 48: 454.
[28] Hiramatsu K. The emergence of Staphylococcus aureus with reduced susceptibility to vancomycin in Japan. Am J Med, 1998, 104: 78.
[29] Zeina B, Greenman J, Purcell WM, et al. Killing of cutaneousm icrobial species by photodynamic therapy. B r J Dermatol, 2002, 146(4):568.
[30] 张红雨，刘为忠．北醌衍生物光敏生物杀菌剂及其制法．中国专利．申请号：021110386．0，2001．
[31] Yoshimura M, Namura S, Akamatsu H, et al. Antimicrobial effects of photo therapy and photochemo therapy in vivo and in vitro. B r J Dermatol, 135:528.
[32] Gasparro FP. Psoralen photobiology: recent advances. Photochem Photobiol, 1996, 63: 553.
[33] Fehr MJ, McCoskey MA, Petrich JW. Light induce dacidification by antiviral agent hypericin. J Am Chem Soc, 1995, 117: 1833.
[34] Lavie G, Mazur Y, Lavie D, et al. Hypericin as an inactivator of infectious viruses in blood components. Transfusion, 1995, 35(5): 392.
[35] North J, Neyndorff H, Levy JG. Photo sensitizers as virucidal agents. J Photochem Photobiol B:Biol, 1993, 17:99.
[36] Nitzan Y, Balzam R, Sudakevitz A, Ashkenazi H. Eradication of Acine to batter baumannii by photo sensitized agents in vitro. J Photochem Photobiol B: Biol, 1998, 42:211.
[37] Saeki Y, Ito Y, Shibata M, et al. Antimicrobial action of green tea extract, flavono flavor and copperchlo rophyll against oral bacteria. Bull Tokyo Dent Coll, 1993, 34(1):33.
[38] 唐蕾，诸葛建．天然光敏剂的光动力抗菌作用．中国抗生素杂志．2004，29(11)：703～706．
[39] 何玉英．科学通报．竹红菌素衍生物在缺氧条件下的光化学．1999，44(22)：2376～2383．
[40] 蒋丽金．竹红菌素的结构、性质、光化学反应及反应机制．科学通报，1990，35(21)：1608～1616．
[41] Chao C, Zhang P. A Facile Route to Peylenquinone. Tetra Lett. 1988, 29(2):225.
[42] Zhao C, Zhang XS, Zhang P. Some Synthetic Studies Related to Peryenequinones. Liebigs Ann Chemt, 1993, 35～41.
[43] 付滨，张傍．天然苝醌合成的初步研究，北京大学学报，2000，36(2)：167-171．
[44] 第五震军，蒋丽金，张曼华．竹红菌甲素光敏氧化反应机制．化学学报，1990，48：483-488．
[45] 朱晓微．贯叶金丝桃研究进展Ⅱ一药代动力学、药效学和临床应用(续)．国外医约·植物药分册，1998，13(5)：210～214．
[46] 李聪，陈远腾，林南英，等．一株产苝醌光敏化合物的真菌成分分析．菌物系统， 2000，19(1)，122～127．
[47] 刘为忠，李聪，谢金伦，等．苝醌类化合物产生菌固态发酵工艺的研究．云南大学学报(自科版)，2000，22(5)：389～391．
[48] Weber T, W elzel K, Pelzer S, et al. Exploiting the genetic potential of polyketide producing strep tomycetes. J Bio technol, 2003, 106 (2-3): 221
[49] 邵荣光，王以光．微生物组合生物合成药物的研究进展与趋势．天津药学．2004，16．(6) 1～3
[50] Ben S. Polyketide biosynthesis beyond the type Ⅰ, Ⅱ and Ⅲ polyketide synthase paradigms. Biocatalysis and biotransformation. 2003, 7: 285～295.
[51] Hopwood DA, Sherman DH. Molecular Genetics of polyketides and its comparison to Fatty acid biosynthesis. Annu Rev Genet, 1990, 24:37～66.
[52] 刘文，唐功利．以生物合成为基础的代谢工程和组合生物合成．中国生物工程杂志．2005(25) 1：1～5
[53] 黄曦，邓子新．聚酮分子生物学进展．国外医约抗生素分册．1998，19(3)：169～176．
[54] Cortes J, Haydock SF, Roberts GA, et al. An unusually large multifunctional polyketide in the erythrornycin—polyketide synthase of Saccharo Polyspora erythraea. Nature. 1990, 346: 176～178.
[55] Donadio S, Stayer MJ, McAlpine JB, Swanson SJ, Katz L. Modular organization of genes required for complex polyketide biosynthesis. Science, 1991, 252: 675～679.
[56] 汪星明，赵凤生．组合生物合成．国外医药抗生素分册．2003，24(2)：49～53．
[57] Nicholson TP, Rudd BA, Dawson M, et al. Design and utility of oligonucleotide gene probes for fungal polyketide synthases. Chem Biol. 2001,8: 157.
[58] Hopwood DA. Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico. Microbiology, 1999, 145:2183.
[59] John K, Borchard T. Genetic engineering may keep one of the richest drug gold mines from being played out. Modern Drag Discovery, 1999, 2:22.
[60] Sambrook J. Molecular Cloning: A laboratory manual, 2nd eds. Cold Spring Harbor Lab Press, 1989, 396～449.
[61] 汤祖辉，赖卫华，许杨．红曲霉RNA的提取方法．江西科学，2002，20(2)：118～120．
[62] 萨姆布鲁克J，弗里奇EF，曼尼阿蒂斯T．分子克隆实验指南(第二版)．北京：科学出版社，1999．343～367．
[63] 李明春，王俊琦，邢来君．丝状真菌深黄被孢霉RNA的提取方法．菌物系统，1999，18(1)：108～111．
[64] 张璘，鱼红闪，金凤燮．真菌中RNA的提取方法．大连轻工业学院学报，2004，23(1)：22～24
[65] 李想，杨奇志，张飞雄．一种小量提取植物总RNA的有效方法．生物技术通报，2004，2：49～51．
[66] Piero C, Catrine K, Akiko K, et al. High-efficiency full-length cDNA cloning by biotinylated CAP trapper, Genomics, 1996, 37:327～336
[67] 朱玉贤、李毅主编．现代分子生物学，北京：高等教育出版社，2002，7：185～189．
[68] Thomas P, Nicholson, Brian AM, et al. Design and utility of oligonucleotide gene probes for fungal polyketide synthases. Chemistry and Biology. 2001, 8(2):157～178.
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