阿维链霉菌与刺糖多孢菌基因组重排筛选高效低毒杀虫剂产生菌的初探
|
摘要
阿维菌素(avermectin)是由阿维链霉菌(Streptomyces avermitilis)产生的大环内酯类抗生素,是迄今为止发现的最有效的杀虫剂,杀螨剂和杀寄生虫剂。多杀菌素( spinosad )是由刺糖多孢菌(Saccharopolyspora spinosa)产生的大环内酯类生物农药,是一种具有触杀及摄食毒性的广谱杀虫剂。本文对阿维菌素和多杀菌素的液相分析方法,阿维链霉菌原生质体的制备和再生,进行了系统性研究,阿维链霉菌与刺糖多孢菌基因组重排技术筛选高效低毒杀虫剂产生菌株进行了初步探索。
建立了一种快速有效的多杀菌素和阿维菌素定量检测方法。采用外标法,以Agilent Zorbax XDB-C18(5μm, 4.6×250 mm)色谱柱,甲醇/乙腈/(含有0.05 %的乙酸铵)水= 45/45/10 (v/ v),作为流动相;流速1.0 mL/min,检测波长246 nm,进样量20μL,柱温30℃。发酵液预处理试验显示:在室温下,采用100 W的超声波处理15 min的超声处理方式,浸提2 h,最适浸提比例为:发酵液(全液):甲醇=1:4。
试验首先对阿维链霉菌的原生质体制备和再生条件进行了研究。考察了种龄、甘氨酸浓度、溶菌酶浓度、溶菌酶作用时间及温度对制备阿维链霉菌原生质体的影响。同时对再生培养基进行了优化,使原生质体再生达到较佳的再生率和生长状态。实验结果现阿维链霉菌在阿维链霉菌在种子培养基YEME中培养40 h至对数生长期,以10%的接种量转接到50 mL 0.5%甘氨酸浓度的YEME培养基中继续培养18 h,收集菌丝体,经1 mg/mL溶菌酶在32℃的水浴条件下震荡水解20 min可获得大量原生质体。
对阿维链霉菌与刺糖多孢菌运用基因重组技术进行育种,筛选高效低毒的重组菌株。通过对ZGD-2进行Co60诱变,原生质体紫外诱变等选育方式得到高产的pop-YUV阿维链霉菌菌株群。pop-YUV与pop-YDS作为出发的亲本菌株进行基因组重排,通过三轮基因组重排最后筛选到了5株菌株,其中GS-4、GS-1阿维菌素比出发菌株ZGD-2产量提高了107.8%,GSD-1多杀菌素产量比出发菌株pop-YDS提高24.4%,JSS-1、JSS-2不产阿维菌素也不产多杀菌,但仍具有杀虫活性。
Avermectins was a novel microbial broad-spectrum insecticide and one of the most effective insecticide,acaricide,parasiticide. Spinosad, which was a novel microbial broad-spectrum insecticide, was secondary metaboli -tes from the aerobic fermentation of a naturally occurring actinomycetes bacterium, Saccharopolyspora spinosa. In this dissertation, the determinati- on of spinosad and avermectin by HPLC, the optimum conditions for preparing and regenerating avermectin producer’-s protoplast,the genome shuffling between Streptomyces avermitilis and Saccharopolyspora spinosa to select the high efficiency and low toxicity strains were studied.
Analyzing method of spinosad by HPLC was introduced. The chromatographic conditions were as follows: Agilent Zorbax XDB-C18 column(5μm,4.6×250mm),mobile phase is Methanol/ acetonitrile/ water= 45/45/10 (v/v).The results also show that flow rate ,detective wavelength, v -olume,the temperature of column were 1.0mL/min,245nm,20μL, 30℃respectively.
The condition for preparation and regenaration of protoplast was optimized .The effects of seed age,the glycin concentration,the lysozyme concentration,the lysozyme treating time and temperature on preparation of protoplast were studied,and the regeneration medium was optimized.A set of media and conditions for formation and regeneration of protolasm was obtained. The result showed that using 2 mg/mL lysozyme at 32℃for 20 mintutes protoplasts were obtained from mycelia of Streptomyces avermitilis which had cultured in a medium cotaining 0.5% glycin.
The research is aim to improve the Streptomyces avermitilis,Sacch- aropolyspora spinosa by genome shuffling and select the high efficiency and low toxicity strains. ZGD-2 was mutanted by Co60 and protoplast’s UV in order to obtain a population called pop-YUV. Then pop-YDS and pop-YUV protoplasts were fused recursively there rounds. The results show that the population of F3, which was obtained after three rounds of protoplast fusion,remarkably improved the uotput. Finally five shuffled strains GS-4,GS-1, GSD-1, JSS-1 and JSS-2.The output of GS-4, GS-1 on avermectin are 107.8% than ZGD-2, and the GSD-1 on spinosad is 24.4% than parent strain .
建立了一种快速有效的多杀菌素和阿维菌素定量检测方法。采用外标法,以Agilent Zorbax XDB-C18(5μm, 4.6×250 mm)色谱柱,甲醇/乙腈/(含有0.05 %的乙酸铵)水= 45/45/10 (v/ v),作为流动相;流速1.0 mL/min,检测波长246 nm,进样量20μL,柱温30℃。发酵液预处理试验显示:在室温下,采用100 W的超声波处理15 min的超声处理方式,浸提2 h,最适浸提比例为:发酵液(全液):甲醇=1:4。
试验首先对阿维链霉菌的原生质体制备和再生条件进行了研究。考察了种龄、甘氨酸浓度、溶菌酶浓度、溶菌酶作用时间及温度对制备阿维链霉菌原生质体的影响。同时对再生培养基进行了优化,使原生质体再生达到较佳的再生率和生长状态。实验结果现阿维链霉菌在阿维链霉菌在种子培养基YEME中培养40 h至对数生长期,以10%的接种量转接到50 mL 0.5%甘氨酸浓度的YEME培养基中继续培养18 h,收集菌丝体,经1 mg/mL溶菌酶在32℃的水浴条件下震荡水解20 min可获得大量原生质体。
对阿维链霉菌与刺糖多孢菌运用基因重组技术进行育种,筛选高效低毒的重组菌株。通过对ZGD-2进行Co60诱变,原生质体紫外诱变等选育方式得到高产的pop-YUV阿维链霉菌菌株群。pop-YUV与pop-YDS作为出发的亲本菌株进行基因组重排,通过三轮基因组重排最后筛选到了5株菌株,其中GS-4、GS-1阿维菌素比出发菌株ZGD-2产量提高了107.8%,GSD-1多杀菌素产量比出发菌株pop-YDS提高24.4%,JSS-1、JSS-2不产阿维菌素也不产多杀菌,但仍具有杀虫活性。
Avermectins was a novel microbial broad-spectrum insecticide and one of the most effective insecticide,acaricide,parasiticide. Spinosad, which was a novel microbial broad-spectrum insecticide, was secondary metaboli -tes from the aerobic fermentation of a naturally occurring actinomycetes bacterium, Saccharopolyspora spinosa. In this dissertation, the determinati- on of spinosad and avermectin by HPLC, the optimum conditions for preparing and regenerating avermectin producer’-s protoplast,the genome shuffling between Streptomyces avermitilis and Saccharopolyspora spinosa to select the high efficiency and low toxicity strains were studied.
Analyzing method of spinosad by HPLC was introduced. The chromatographic conditions were as follows: Agilent Zorbax XDB-C18 column(5μm,4.6×250mm),mobile phase is Methanol/ acetonitrile/ water= 45/45/10 (v/v).The results also show that flow rate ,detective wavelength, v -olume,the temperature of column were 1.0mL/min,245nm,20μL, 30℃respectively.
The condition for preparation and regenaration of protoplast was optimized .The effects of seed age,the glycin concentration,the lysozyme concentration,the lysozyme treating time and temperature on preparation of protoplast were studied,and the regeneration medium was optimized.A set of media and conditions for formation and regeneration of protolasm was obtained. The result showed that using 2 mg/mL lysozyme at 32℃for 20 mintutes protoplasts were obtained from mycelia of Streptomyces avermitilis which had cultured in a medium cotaining 0.5% glycin.
The research is aim to improve the Streptomyces avermitilis,Sacch- aropolyspora spinosa by genome shuffling and select the high efficiency and low toxicity strains. ZGD-2 was mutanted by Co60 and protoplast’s UV in order to obtain a population called pop-YUV. Then pop-YDS and pop-YUV protoplasts were fused recursively there rounds. The results show that the population of F3, which was obtained after three rounds of protoplast fusion,remarkably improved the uotput. Finally five shuffled strains GS-4,GS-1, GSD-1, JSS-1 and JSS-2.The output of GS-4, GS-1 on avermectin are 107.8% than ZGD-2, and the GSD-1 on spinosad is 24.4% than parent strain .
引文
[1]扈洪波,朱蓓蕾,李俊锁.阿维菌素类药物研究进展[J].畜牧兽医学报, 2000, 31(6): 520-529
[2]Dutton CJ Gibson SP, Goudie AC, Holdom KS,et al.Novel avermectins produced by mutation biosynthesis[J]. Antibiot, 1991, 44(3): 357～365
[3]Gandecha AR, Large SL, Cundliffe E. Analysis of four tylosin biosynthetic genes from the tyiLM region of the Streptomyces fradiae genome[J]. Gene,l997.184(2):197～203.
[4]Burg RW, Miller BM, Baker EE, et al. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation[J]. Antimicrob Agents Chemother, 1979, 15, 361-367.
[5]Korystov YN, Ermakova NV, Kublik LN, et al. Avermectins inhibit the multidrug resistance of tumor cells[J]. Eur Pharmacol, 2004, 493, 57-64.
[6] Geary, TG.. Ivermectin 20 years on: maturation of a wonder drug[J]. Trends Parasitol, 2005, 21, 530-532.
[7]Lespine A, Dupuy J, Orlowski S, et al. Interaction of ivermectin with multidrug resistance proteins (MRP1, 2and 3) [J]. Chem Biol Interact, 2006, 159, 169-179.
[8]Lespine A, Martin S, Dupuy J, et al. Interaction of macrocyclic lactones with P-glycoprotein: structure-affinity relationship[J]. Eur Pharmacol Sci, 2007, 30, 84-94.
[9]Ikeda H, Nonomiya T,ōmura S. Organization of biosynthetic gene cluster for averm ectin in Str -eptomyces avermitilis:analysis of enzymatic domains in four polyketide synthases[J]. Ind Microbiol Biotechnol, 2001, 27: 170–176.
[10]Burg R W, Willer E E, et al, Avermectins, new family of potent anthelmintie agents:producingorganisms and femenntation[J] . Antimicrob Agent Chem.1979, 15: 361-367
[11]张卫,林匡飞等阿维菌素在土壤中的光解研究[J] .农业环境科学学报2006, 25(3): 741-744
[12]Shoop W L, Morzik H, Fisher M H. St ructure and activity of avermectins and milbemycins In animal health[J ]. Veterinary Parasitology, 1995, 59, 139～156.
[13]Turner M J,Schaeffer J M. Mode of action of ivermectin[M]. In Ivermectin and Abamectin. Ed. Cam pbell W.C.pp.Springer - Verlag, New York. 1989, 73～88.
[14]Shoop W L, Mrozik H, Fisher M H. Structure and activity of avermectins and milbemy cins in animal health[J ].Veterinry Parasitology, 1995, 59(2): 139 - 156.
[15]Campbell W C, Benz G W.Ivermectin: a review of efficacy and safety[J ]. Journal of Veterinar -y Pharmacology and Therapeutics, 1984, 7: 1-16.
[16]Shoop W L,Haines H Wmichael B F.Mutualresistance between avermectins and milbermycin -s:oral activity of ivermectin and moxidectin against ivermectin resistance and susceptible nematodes [J]. The Veterinary Record,1993, 133: 445 - 447.
[17]MekelarQ A, BenchaouiH A.. Avermectins andmilbermycins[J]. Journal of Veterinary Pharmac -ology andTherapeutics, 1996, 19: 331– 351.
[18]汪晓飞.藏野驴阿维菌素中毒的抢救[J ].青海畜牧兽医杂志, 2000, 4 (2): 22.
[19]Ikeda H, Takada Y, Pang CH, Tanaka H,ōmura S, Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis[J]. Bacterio, 1993, l175: 2077-2082.
[20]keda H, Nonomiya T, Usami M, Ohta T,ōmura S.Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis[J]. Proc Natl Acad Sci USA, 1999, 96: 9509–9514.
[21]Macneil T, Gewain KM, MacNeil DJ Deletion analysis of the avermectin biosynthetic genes of Streptomyces avermitilis by gene cluster displacement[J]. Bacteriol 1993,175:2552–2563
[22]Ikeda H, Nonomiya T, Usami M, Ohta T,ōmura S.Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis[J]. Proc Natl Acad Sci USA, 1999, 96: 9509–9514.
[23]Ikeda H, Idhikawa J, Hanamoto A,et al. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis[J].Nat Biotechnol.2003,21:526-531.
[24]Cane DE, Liang TC, Kaplan L, etal.Biosynthetic origin of the carbon skeleton and oxygenatoms of the avermectins[J]. Am Chem Soc, 1983, 105: 4110–4112.
[25]Omura Satoshi,I keda Haruo, etal. Selective Production of Specific Component of Avermectins in streptomyces avermitilis[J]. 1991,Antibiot 44(5): 560-563.
[26]Skinner DD, Morgenstern MR, Fedechko RW, Denoya CD.Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alphabeta] component in Escherichia coli[J]. Bacteriol, 1995, 177: 183–190.
[27]Denoya CD, Fedechko RW, Hafner EW, etal.A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins[J]. Bacteriol, 1995, 177: 3504–3511.
[28]Dutton C J,Gibson S P,Goudie A C, et al.Novel avermectins produced by mutational biosynthesis[J]. Antibiot(Tokyo) , 1991, 44(3): 357.
[29]Matsuoka Y, Okazaki M, Kitamura Y, et al. Developmental expression of P-glycoprotein (m -ultidrug resistance gene product) in the rat brain [J]. Neurobiol, 1999, 39: 383-392.
[30]Pang CH.Matsuzaki K.Ikeda H.Production of a new methylated 6,8a-seo-6, 8a–deoxy derivative of the avermectins bu a transformant strain of Streptomyces avermilitis[J]. Antibio -t,1995,48(1): 93-94.
[31]Ikeda H,Pang CH,Endo H.Construction of a single component producerfrom the widl type avermentin producer Sreptomyces avermitilis[J]. Antibiot.1995a,48(6):532-534 ).
[32]Ikeda H, Kotaki H,ōmura S.Genetic studies of avermectin biosynthesis in Streptomyces avermitilis[J].Bacteriol,1987,169:5615–5621.
[33]Victor A. Drinyaev, Vladimir A.Mosin, et al. Antitumor effect avermectins[J]. Eur Pharmacol, 2004, 501: 19-23.
[34]Juliano RL, LingV. Surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants[J]. Biophys Acta, 1976, 455, (1): 152-162.
[35]Stouch TR, GudmundssonO.Progressin understanding the structure activity relationships of P-glycoprotein[J]. Adv Drug Del Rev, 2002, 54 (3): 315-3281.
[36]Azzaria am, Schurr E. Discrete mutati on introduced in the predicted nucleotide binding sites of the MDR1 gene abolished its ability to confer mutidrug resistance[J]. Mol Cell Biol, 1989, 9:5289-5297.
[37]Loscher W, Potschka H. Blood-brain barrier active efflux transporters: ATP-binding cassette gene family[J]. NeuroRx, 2005, 2: 86-98.
[38]Kwei GY, Alvaro RF, Chen Q, et al. Disposition of ivermectin and cyclosporin A in CF-1 mice deficient in mdr1a P-glycoprot-ein[J]. Drug Metab Dispos, 1999, 27: 581-587.
[39]Schinkel AH, Smit JJ, van TellingenO, et al. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs [J]. Cell, 1994, 77(4): 491-502.
[40]Schinkel AH, Wagenaar E, et al. Absence of the mdr1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin and cyclosporin A [J]. Clin In vest, 1995, 96 (4): 1698-1705.
[41]Schinkel AH, Wagenaar E, Mol CA, et al. P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs [J]. J Clin Invest, 1996, 97(11): 2517-2524.
[42]Lankas GR, Cartwright ME, Umbenhauer D. P-Glycoprotein deficiency in a subpopulation of CF-1 mice enhances avermectin induced neurotoxicity[J]. Toxicol Appl Pharmacol, 1997, 143: 357-365.
[43]Umbenhauer DR, Lankas GR, Pippert TR, et al. Identiffication of a P-glycoprotein-deficient subpopulation in the CF-1 mouse strain using a restriction fragment length polymorphism[J]. Toxicol Appl Pharmacol, 1997, 146: 88-94.
[44]Lankas GR, Wise LD, Cartwright ME, et al. Placental P-glycoprotein deficiency enhances susceptibility to chemically induced birth defects in mice[J]. Reprod Toxicol, 1998, 12: 457-463.
[45]Matsuoka Y, Okazaki M, Kitamura Y, et al. Developmental expression of P-glycoprotein 0(multidrug resistance gene product) in the rat brain [J]. Neurobiol, 1999, 39: 383-392.
[46]Lankas GR, Minsker DH, Robertson RT. Effects of ivermectin on reproduction and neonatal toxicity in rats [J]. Food Chem Toxico, 1989, l27: 523-529.
[47]Ying-xin Zhang, Kim Perry, Victor A.Vinci, et al. Genome shuffling leads to rapid phenotypic improvement in bacteria [J]. Nature. 2002, 15: 644-646.
[1]李歆,李小玲.高效液相色谱技术在饲料安全生产中的应用[J].畜牧与饲料科学, 2009, 30(1): 92-94.
[2] David C Woollard, Harvey E Indyk, Scott K Christiansen. The analysis of pantothenic acid in milk and infant formulas by HPLC[J]. Food Chemistr, 2000, 69: 201-208.
[3]龚炳永,周亦昌.抗生素的分离与测定[J].色谱, 1992, 10(5): 269-277.
[4] Duncan E S, Alessio C, Chris A. Coenzyme biosynthesis: enzyme mechanism, structure and inhibition[J]. Nat Prod Rep, 2007, 24: 1009–1026.
[5]聂果,王广成,张忠明,等.多杀菌素高效液相色谱分析[J].农药科学与管理, 2003, 24(8): 6-8.
[6]陈小龙,郑裕国,沈寅初.农用抗生素多杀菌素的研究进展[J].农药, 2002, 41(1): 4-7.
[7]张苑,金志华.多杀菌素的高效液相色谱[J].农药, 2003, 42(10): 27-28.
[8]吴春先,聂果.高效液相色谱法测定水中多杀菌素的残留量[J].农药, 2006, 45(3): 191-193.
[1]施巧琴,吴松刚.工业微生物育种学[M].北京:科学出版社, 2003: 290-295.
[2]赵凯,周东坡,平文祥,马玺.产紫杉醇菌株原生质体诱变育种的研究[J].生物工程学报, 2005, 21(5): 848-851
[3]徐波,王明蓉,夏永等.应用基因组重排育种新方法筛选替考拉宁高产菌[J].中国抗生素志, 2006, 31(4): 237-242 7
[4]徐志南,董悦涵,谢志鹏,岑沛霖.米多霉素生产菌原生质体融合研究[J].浙江大学学报, 2006, 40(7): 1262-126
[5] Dai M, Copley SD, Genome shuffling improves degradation of the anthropogenic pesticide Pentachllorophenol by Sphingbium chlorophenolicum ATCC39723[J]. Appl Environ Microbiol, 2004, 70(4): 2391-2397.
[6]陈涛,王靖宇,周世奇等.基因组改组及代谢通量分析在产核黄素Bacilus subtilis性能改进中的应用[J].化工学报, 2004, 55(11): 1842-1848.
[7]朱慧.纳他霉素产生菌基因组重排育种[D].浙江大学, 2006.
[8]林塞珍.多杀菌素产生菌基因组重排育种研究[D].浙江大学, 2007.
[9]朱林东.普那霉素产生菌基因组重排育种[D].浙江大学, 2007.
[10]沈文和.雷帕霉素高产菌株的选育及培养条件的优化[D].浙江大学, 2006.
[1]郭伟群.阿维菌素高产菌株的选育和发酵条件的优化[D].中国农业大学, 2007.
[2]朱慧.纳他霉素产生菌基因组重排育种[D].浙江大学, 2006.
[3]戴伟国.中国阿维菌素的前景展望[J].中国制药信息, 2004, 20(9): 33-35.
[4]曹友声,刘仲敏.现代工业微生物学[M],长沙:湖南科学技术出版社. 1998.
[5]Olga S, Fernando , Volker. S. Rapid evolution of novel strains in microorganisms[J]. A pplied a -nd Environmental Microbiology.2001, 67(8): 3645-3649.
[6]Yasuyo O, Masako A, Zeno M. The Iinfluence of Histidine on the Color of Cu-complex Azodyes -on Cellulose[J]. Dyes and Pigments, 1996, 31 (1): 53-67.
[7] Burg R W, Miller B M, Baker E E, et al. Avermectins, new family of potent anthelmintic agen- ts: Producing organisms and fermentationn[J].Antimicrob Agents Chemother, 1979, 15: 361-367.
[8]Pinna L A, Lorini M, Moret V, et al. Effect of oligomycin and sucicinate on mitoch-ondrial metabolism of adenine nucleotides[J]. Biochim Biophys Acta, 1967, 143: 18-25.
[1]ZhangY X, Perry K, Vinci VA.Genome Shuffling leads to rapid phenotypic improvement in bacteria[J]. Nature, 2002, 15, 664-646.
[2]Patnaik R, Louie S, Gavrilovie V. Genome Shuffling of Lactobacillus for Improved Acid Toleran- ce[J]. Nature Biotechnology, 2002, 20, 707-712.
[3]Atephanopoulos G. Metabolic engineering by genome shuffling[J] .Nature Biotechology 2002, 20 (7), 666-668.
[4]朱东林,普纳霉素产生菌基因组重排[D].浙江大学,金志华,浙江杭州, 2006.
[5]朱慧.纳他霉素产生菌基因组重排育种[D].浙江大学, 2006.
[6]沈文和.雷帕霉素高产菌株的选育及培养条件的优化[D].浙江大学, 2006.
[2]Dutton CJ Gibson SP, Goudie AC, Holdom KS,et al.Novel avermectins produced by mutation biosynthesis[J]. Antibiot, 1991, 44(3): 357～365
[3]Gandecha AR, Large SL, Cundliffe E. Analysis of four tylosin biosynthetic genes from the tyiLM region of the Streptomyces fradiae genome[J]. Gene,l997.184(2):197～203.
[4]Burg RW, Miller BM, Baker EE, et al. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation[J]. Antimicrob Agents Chemother, 1979, 15, 361-367.
[5]Korystov YN, Ermakova NV, Kublik LN, et al. Avermectins inhibit the multidrug resistance of tumor cells[J]. Eur Pharmacol, 2004, 493, 57-64.
[6] Geary, TG.. Ivermectin 20 years on: maturation of a wonder drug[J]. Trends Parasitol, 2005, 21, 530-532.
[7]Lespine A, Dupuy J, Orlowski S, et al. Interaction of ivermectin with multidrug resistance proteins (MRP1, 2and 3) [J]. Chem Biol Interact, 2006, 159, 169-179.
[8]Lespine A, Martin S, Dupuy J, et al. Interaction of macrocyclic lactones with P-glycoprotein: structure-affinity relationship[J]. Eur Pharmacol Sci, 2007, 30, 84-94.
[9]Ikeda H, Nonomiya T,ōmura S. Organization of biosynthetic gene cluster for averm ectin in Str -eptomyces avermitilis:analysis of enzymatic domains in four polyketide synthases[J]. Ind Microbiol Biotechnol, 2001, 27: 170–176.
[10]Burg R W, Willer E E, et al, Avermectins, new family of potent anthelmintie agents:producingorganisms and femenntation[J] . Antimicrob Agent Chem.1979, 15: 361-367
[11]张卫,林匡飞等阿维菌素在土壤中的光解研究[J] .农业环境科学学报2006, 25(3): 741-744
[12]Shoop W L, Morzik H, Fisher M H. St ructure and activity of avermectins and milbemycins In animal health[J ]. Veterinary Parasitology, 1995, 59, 139～156.
[13]Turner M J,Schaeffer J M. Mode of action of ivermectin[M]. In Ivermectin and Abamectin. Ed. Cam pbell W.C.pp.Springer - Verlag, New York. 1989, 73～88.
[14]Shoop W L, Mrozik H, Fisher M H. Structure and activity of avermectins and milbemy cins in animal health[J ].Veterinry Parasitology, 1995, 59(2): 139 - 156.
[15]Campbell W C, Benz G W.Ivermectin: a review of efficacy and safety[J ]. Journal of Veterinar -y Pharmacology and Therapeutics, 1984, 7: 1-16.
[16]Shoop W L,Haines H Wmichael B F.Mutualresistance between avermectins and milbermycin -s:oral activity of ivermectin and moxidectin against ivermectin resistance and susceptible nematodes [J]. The Veterinary Record,1993, 133: 445 - 447.
[17]MekelarQ A, BenchaouiH A.. Avermectins andmilbermycins[J]. Journal of Veterinary Pharmac -ology andTherapeutics, 1996, 19: 331– 351.
[18]汪晓飞.藏野驴阿维菌素中毒的抢救[J ].青海畜牧兽医杂志, 2000, 4 (2): 22.
[19]Ikeda H, Takada Y, Pang CH, Tanaka H,ōmura S, Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis[J]. Bacterio, 1993, l175: 2077-2082.
[20]keda H, Nonomiya T, Usami M, Ohta T,ōmura S.Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis[J]. Proc Natl Acad Sci USA, 1999, 96: 9509–9514.
[21]Macneil T, Gewain KM, MacNeil DJ Deletion analysis of the avermectin biosynthetic genes of Streptomyces avermitilis by gene cluster displacement[J]. Bacteriol 1993,175:2552–2563
[22]Ikeda H, Nonomiya T, Usami M, Ohta T,ōmura S.Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis[J]. Proc Natl Acad Sci USA, 1999, 96: 9509–9514.
[23]Ikeda H, Idhikawa J, Hanamoto A,et al. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis[J].Nat Biotechnol.2003,21:526-531.
[24]Cane DE, Liang TC, Kaplan L, etal.Biosynthetic origin of the carbon skeleton and oxygenatoms of the avermectins[J]. Am Chem Soc, 1983, 105: 4110–4112.
[25]Omura Satoshi,I keda Haruo, etal. Selective Production of Specific Component of Avermectins in streptomyces avermitilis[J]. 1991,Antibiot 44(5): 560-563.
[26]Skinner DD, Morgenstern MR, Fedechko RW, Denoya CD.Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alphabeta] component in Escherichia coli[J]. Bacteriol, 1995, 177: 183–190.
[27]Denoya CD, Fedechko RW, Hafner EW, etal.A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins[J]. Bacteriol, 1995, 177: 3504–3511.
[28]Dutton C J,Gibson S P,Goudie A C, et al.Novel avermectins produced by mutational biosynthesis[J]. Antibiot(Tokyo) , 1991, 44(3): 357.
[29]Matsuoka Y, Okazaki M, Kitamura Y, et al. Developmental expression of P-glycoprotein (m -ultidrug resistance gene product) in the rat brain [J]. Neurobiol, 1999, 39: 383-392.
[30]Pang CH.Matsuzaki K.Ikeda H.Production of a new methylated 6,8a-seo-6, 8a–deoxy derivative of the avermectins bu a transformant strain of Streptomyces avermilitis[J]. Antibio -t,1995,48(1): 93-94.
[31]Ikeda H,Pang CH,Endo H.Construction of a single component producerfrom the widl type avermentin producer Sreptomyces avermitilis[J]. Antibiot.1995a,48(6):532-534 ).
[32]Ikeda H, Kotaki H,ōmura S.Genetic studies of avermectin biosynthesis in Streptomyces avermitilis[J].Bacteriol,1987,169:5615–5621.
[33]Victor A. Drinyaev, Vladimir A.Mosin, et al. Antitumor effect avermectins[J]. Eur Pharmacol, 2004, 501: 19-23.
[34]Juliano RL, LingV. Surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants[J]. Biophys Acta, 1976, 455, (1): 152-162.
[35]Stouch TR, GudmundssonO.Progressin understanding the structure activity relationships of P-glycoprotein[J]. Adv Drug Del Rev, 2002, 54 (3): 315-3281.
[36]Azzaria am, Schurr E. Discrete mutati on introduced in the predicted nucleotide binding sites of the MDR1 gene abolished its ability to confer mutidrug resistance[J]. Mol Cell Biol, 1989, 9:5289-5297.
[37]Loscher W, Potschka H. Blood-brain barrier active efflux transporters: ATP-binding cassette gene family[J]. NeuroRx, 2005, 2: 86-98.
[38]Kwei GY, Alvaro RF, Chen Q, et al. Disposition of ivermectin and cyclosporin A in CF-1 mice deficient in mdr1a P-glycoprot-ein[J]. Drug Metab Dispos, 1999, 27: 581-587.
[39]Schinkel AH, Smit JJ, van TellingenO, et al. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs [J]. Cell, 1994, 77(4): 491-502.
[40]Schinkel AH, Wagenaar E, et al. Absence of the mdr1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin and cyclosporin A [J]. Clin In vest, 1995, 96 (4): 1698-1705.
[41]Schinkel AH, Wagenaar E, Mol CA, et al. P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs [J]. J Clin Invest, 1996, 97(11): 2517-2524.
[42]Lankas GR, Cartwright ME, Umbenhauer D. P-Glycoprotein deficiency in a subpopulation of CF-1 mice enhances avermectin induced neurotoxicity[J]. Toxicol Appl Pharmacol, 1997, 143: 357-365.
[43]Umbenhauer DR, Lankas GR, Pippert TR, et al. Identiffication of a P-glycoprotein-deficient subpopulation in the CF-1 mouse strain using a restriction fragment length polymorphism[J]. Toxicol Appl Pharmacol, 1997, 146: 88-94.
[44]Lankas GR, Wise LD, Cartwright ME, et al. Placental P-glycoprotein deficiency enhances susceptibility to chemically induced birth defects in mice[J]. Reprod Toxicol, 1998, 12: 457-463.
[45]Matsuoka Y, Okazaki M, Kitamura Y, et al. Developmental expression of P-glycoprotein 0(multidrug resistance gene product) in the rat brain [J]. Neurobiol, 1999, 39: 383-392.
[46]Lankas GR, Minsker DH, Robertson RT. Effects of ivermectin on reproduction and neonatal toxicity in rats [J]. Food Chem Toxico, 1989, l27: 523-529.
[47]Ying-xin Zhang, Kim Perry, Victor A.Vinci, et al. Genome shuffling leads to rapid phenotypic improvement in bacteria [J]. Nature. 2002, 15: 644-646.
[1]李歆,李小玲.高效液相色谱技术在饲料安全生产中的应用[J].畜牧与饲料科学, 2009, 30(1): 92-94.
[2] David C Woollard, Harvey E Indyk, Scott K Christiansen. The analysis of pantothenic acid in milk and infant formulas by HPLC[J]. Food Chemistr, 2000, 69: 201-208.
[3]龚炳永,周亦昌.抗生素的分离与测定[J].色谱, 1992, 10(5): 269-277.
[4] Duncan E S, Alessio C, Chris A. Coenzyme biosynthesis: enzyme mechanism, structure and inhibition[J]. Nat Prod Rep, 2007, 24: 1009–1026.
[5]聂果,王广成,张忠明,等.多杀菌素高效液相色谱分析[J].农药科学与管理, 2003, 24(8): 6-8.
[6]陈小龙,郑裕国,沈寅初.农用抗生素多杀菌素的研究进展[J].农药, 2002, 41(1): 4-7.
[7]张苑,金志华.多杀菌素的高效液相色谱[J].农药, 2003, 42(10): 27-28.
[8]吴春先,聂果.高效液相色谱法测定水中多杀菌素的残留量[J].农药, 2006, 45(3): 191-193.
[1]施巧琴,吴松刚.工业微生物育种学[M].北京:科学出版社, 2003: 290-295.
[2]赵凯,周东坡,平文祥,马玺.产紫杉醇菌株原生质体诱变育种的研究[J].生物工程学报, 2005, 21(5): 848-851
[3]徐波,王明蓉,夏永等.应用基因组重排育种新方法筛选替考拉宁高产菌[J].中国抗生素志, 2006, 31(4): 237-242 7
[4]徐志南,董悦涵,谢志鹏,岑沛霖.米多霉素生产菌原生质体融合研究[J].浙江大学学报, 2006, 40(7): 1262-126
[5] Dai M, Copley SD, Genome shuffling improves degradation of the anthropogenic pesticide Pentachllorophenol by Sphingbium chlorophenolicum ATCC39723[J]. Appl Environ Microbiol, 2004, 70(4): 2391-2397.
[6]陈涛,王靖宇,周世奇等.基因组改组及代谢通量分析在产核黄素Bacilus subtilis性能改进中的应用[J].化工学报, 2004, 55(11): 1842-1848.
[7]朱慧.纳他霉素产生菌基因组重排育种[D].浙江大学, 2006.
[8]林塞珍.多杀菌素产生菌基因组重排育种研究[D].浙江大学, 2007.
[9]朱林东.普那霉素产生菌基因组重排育种[D].浙江大学, 2007.
[10]沈文和.雷帕霉素高产菌株的选育及培养条件的优化[D].浙江大学, 2006.
[1]郭伟群.阿维菌素高产菌株的选育和发酵条件的优化[D].中国农业大学, 2007.
[2]朱慧.纳他霉素产生菌基因组重排育种[D].浙江大学, 2006.
[3]戴伟国.中国阿维菌素的前景展望[J].中国制药信息, 2004, 20(9): 33-35.
[4]曹友声,刘仲敏.现代工业微生物学[M],长沙:湖南科学技术出版社. 1998.
[5]Olga S, Fernando , Volker. S. Rapid evolution of novel strains in microorganisms[J]. A pplied a -nd Environmental Microbiology.2001, 67(8): 3645-3649.
[6]Yasuyo O, Masako A, Zeno M. The Iinfluence of Histidine on the Color of Cu-complex Azodyes -on Cellulose[J]. Dyes and Pigments, 1996, 31 (1): 53-67.
[7] Burg R W, Miller B M, Baker E E, et al. Avermectins, new family of potent anthelmintic agen- ts: Producing organisms and fermentationn[J].Antimicrob Agents Chemother, 1979, 15: 361-367.
[8]Pinna L A, Lorini M, Moret V, et al. Effect of oligomycin and sucicinate on mitoch-ondrial metabolism of adenine nucleotides[J]. Biochim Biophys Acta, 1967, 143: 18-25.
[1]ZhangY X, Perry K, Vinci VA.Genome Shuffling leads to rapid phenotypic improvement in bacteria[J]. Nature, 2002, 15, 664-646.
[2]Patnaik R, Louie S, Gavrilovie V. Genome Shuffling of Lactobacillus for Improved Acid Toleran- ce[J]. Nature Biotechnology, 2002, 20, 707-712.
[3]Atephanopoulos G. Metabolic engineering by genome shuffling[J] .Nature Biotechology 2002, 20 (7), 666-668.
[4]朱东林,普纳霉素产生菌基因组重排[D].浙江大学,金志华,浙江杭州, 2006.
[5]朱慧.纳他霉素产生菌基因组重排育种[D].浙江大学, 2006.
[6]沈文和.雷帕霉素高产菌株的选育及培养条件的优化[D].浙江大学, 2006.