红树林土壤总DNA不同提取方法比较研究以及Ⅰ型聚酮合成酶基因检测
|
摘要
获得高浓度、大片段、无偏好的土壤总DNA是土壤微生物群落结构研究和宏基因组文库构建的基础。本研究采用了8种方法从红树林土壤中提取DNA,并对8种方法提取出的DNA的质量和产量进行比较评价。结果表明,8种方法均可从土壤中提取到DNA,但不同方法提取到DNA的产量和质量存在明显差异。Bio 101 FastPrep? SPIN Kit (for Soil)抽提到的DNA得率最高,适合分子生态学研究;细胞包埋法提取的DNA纯度最高,所得到的DNA片段最大(>48kb),有利于构建大分子量DNA宏基因组文库,充分利用微生物多样性资源开发新活性物质和基因;SDS-GITC- PEG法可提取出较高质量和产量的DNA,且DNA片段较大(>23kb),可用于构建小分子量宏基因组文库,筛选小分子酶类。而SDS-GITC- FastPrep?- PEG法与Bio 101 Kit相比,价格便宜,适合于广大普通实验室进行土壤DNA提取工作。
用I型聚酮合成酶基因特异引物对提取出的红树林土壤DNA进行PCR扩增,电泳结果显示被测试的五个土壤样品均有特异性条带。由此推测本实验所采集的红树林土壤样品总DNA中存在有I型聚酮合成酶基因,即这几种红树林土壤中可能含有I型聚酮类天然产物产生菌。
Isolation of DNA sufficient in purity, large in fragment and unbiased representation of the microbial diversity from natural environments is the basis for molecular ecology study on soil microbial community structure. In this experiment, eight different methods commonly used to extract DNA from soil were employed to extract DNA from mangrove soil. Quality and quantity of the extracted DNA were evaluated for the eight methods. The results showed that all of the eight methods could obtain the soil DNA, but the quality and quantity were different. Bio 101 FastPrep? SPIN Kit (for Soil) could obtain the highest quantity of soil DNA and can be used for microbial community investigation. The method using Cell Embed produce the purest DNA, and the DNA fragments are largest in size (>48kb) suitable for construct a high molecular weight metagenome library, exploit the most of microbial diversity resource to find novel bioactive compounds or genes. SDS-GITC- PEG method could obtain the higher quality and quantity of soil DNA(>23kb), and can be used to construct a low molecular weight metagenome library for enzyme screening. SDS-GITC-FastPrep?-PEG method was feasible for most labs to extract soil DNA for its low cost.
The DNA extracted from five mangrove soil samples were amplified by PCR using the specific primers for type I polyketide synthetase gene. The electrophoresis results showed that all the five soil samples appeared the typical band of type I polyketide synthetase gene. It is indicated that there were type I polyketide synthetase genes, thus, there must be microorganisms that can synthesis type I polyketide compounds in these mangrove soils.
用I型聚酮合成酶基因特异引物对提取出的红树林土壤DNA进行PCR扩增,电泳结果显示被测试的五个土壤样品均有特异性条带。由此推测本实验所采集的红树林土壤样品总DNA中存在有I型聚酮合成酶基因,即这几种红树林土壤中可能含有I型聚酮类天然产物产生菌。
Isolation of DNA sufficient in purity, large in fragment and unbiased representation of the microbial diversity from natural environments is the basis for molecular ecology study on soil microbial community structure. In this experiment, eight different methods commonly used to extract DNA from soil were employed to extract DNA from mangrove soil. Quality and quantity of the extracted DNA were evaluated for the eight methods. The results showed that all of the eight methods could obtain the soil DNA, but the quality and quantity were different. Bio 101 FastPrep? SPIN Kit (for Soil) could obtain the highest quantity of soil DNA and can be used for microbial community investigation. The method using Cell Embed produce the purest DNA, and the DNA fragments are largest in size (>48kb) suitable for construct a high molecular weight metagenome library, exploit the most of microbial diversity resource to find novel bioactive compounds or genes. SDS-GITC- PEG method could obtain the higher quality and quantity of soil DNA(>23kb), and can be used to construct a low molecular weight metagenome library for enzyme screening. SDS-GITC-FastPrep?-PEG method was feasible for most labs to extract soil DNA for its low cost.
The DNA extracted from five mangrove soil samples were amplified by PCR using the specific primers for type I polyketide synthetase gene. The electrophoresis results showed that all the five soil samples appeared the typical band of type I polyketide synthetase gene. It is indicated that there were type I polyketide synthetase genes, thus, there must be microorganisms that can synthesis type I polyketide compounds in these mangrove soils.
引文
1. 董晓毅,王梁华,焦炳华. 聚酮的异源生物合成. 厦门, 第三届海洋生物高科技论坛,2005,19～23.
2. 林鹏 中国红树林研究进展 厦门大学学报(自然科学版) 2001, 40(2): 592～603.
3. 腾 应, 骆永明, 赵祥伟, 李振高, 宋 静, 吴龙华. 重金属复合污染农田土壤DNA 的快速提取及其 PCR- DGGE 分析. 土壤学报, 2004, 41 (3): 343～348.
4. 阎 冰, 洪 葵, 许 云, 马 超. 宏基因组克隆——微生物活性物质筛选的新途径. 微生物学通报, 2005, 32(1): 113～117.
5. 张瑞福,曹慧,崔中利,李顺鹏,樊奔. 土壤微生物总 DNA 的提取和纯化. 微生物学报,2003,43(2):276～282.
6. Andrew E. Berry, Claudia Chiocchini, Tina Selby, Margherita Sosio, Elizabeth M.H Wellington. Isolation of high molecular weight DNA from soil for cloning into BAC vectors. FEMS Microbiology Letters. 2003 ,223: 15～20.
7. Amann R I, Ludwig W, Schleifer K H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev, 1995, 59 (1): 143～169.
8. Arslanian R L, Tang L, Blough S, Ma W, Qiu R G, Katz L, Carney J R. A new cytotoxic epothilone from modified polyketide synthases heterologously expressed in Myxococcus xanthus. J Nat Products, 2002, 65(7): 1061～1064.
9. Bakken L.R. Separation and purification of bacteria from soil. Appl. Environ. Microbiol. 1985, 49: 1482~1487.
10. Bakken L.R., Lindahl V Nucleic Acids in the Environment: Methods and Applications, pringer-Verlag, Heidelberg. Germany, 1995: 9~27.
11. Bedford D J, Schweizer E, Hopwood D A, Khosla C. Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor. J Bacteriol, 1995, 177(15): 4544～4548.
12. Berry A E, Chiocchini C, Selby T, Sosio M, Wellington E M. Isolation of high molecular weight DNA from soil for cloning into BAC vectors. FEMS Microbiol Lett, 2003, 223(1): 15～20.
13. Bertrand H, Poly F, Van VT, Lombard N, Nalin R, Vogel TM, Simonet P. High molecular weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction. J Microbiol Meth, 2005, 62(1): 1～11.
14. Burgmann H., Pesaro M., Widmer F., Zeyer J. A strategy for optimizing quality and quantity of DNA extracted from soil, J. Microbiol. Methods, 2001, 45: 7~20.
15. Chandler D.P., Frederickson J.K., Brockman J. Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries. Mol. Ecol. 1997, 6: 475~482.
16. Courtois S., Frostegard A., Goransson P., Depret G., Jeannin P., Simonet P. Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation, Environ.Microbiol. 2001, 3: 431~439.
17. Courtois S, Cappellano CM, Ball M, Francou F-X, Normand P, Helynck G, Martinez A, Kolvek SJ, Hopke J, Osburne MS. Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products. Appl. Environ. Microbiol. 2003, 69: 49–55.
18. Cullen D. W., Hirsch P. R. Simple and rapid method for direct extraction of microbial DNA from soil for PCR. Soil Biol.Biotech, 1998, 30: 983～993.
19. Dojka MA, Harris JK, Pace NR. Expanding the known diversity and environmental distribution of an unculture phylogenetic division of bacteria. Environ. Microbiol. 2000, 66: 1617-1621.
20. Entcheva P, Liebl W, Johann A, HartschT, Streit WR. Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia. Appl. Environ.Microbiol. 2001, 67:89–99
21. Elsas J D, Duarte G F, Rosado A S. Microbiological and molecular biological methods for monitoring microbial inoculants and their effects in the soil environment. Jour Microbial Methods. 1998, 32 : 133～154.
22. Faegri A., Torsvik V.L., Goksoyr J. Bacterial and fungal activities in soil: separation of bacteria and fungi by a rapid fractionated centrifugation technique, Soil Biol. Biochem. 1977, 9: 105~112.
23. Fernandez-Moreno M A, Martinez E, Boto L, Hopwood DA, Malpartida F. Nucleotide sequence and deduced functions of a set of cotranscribed genes of S t reptomyces coelicolor A 3 (2) including the polyketide synthase for the antibiotic actinorhodin. J Biol Chem, 1992, 267(27):19278～19290.
24. Fu H, Ebert-Khosla S, Hopwood D A, Khosla C. Relaxed specificity of the oxytetracycline polyketide synthase for an acetate primer in the absence of a malonyamyl primer. J Am Chem Soc, 1994, 116(14): 6443～6444.
25. Frostegard A, Courtois S, Ramisse V, et al. Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbio.1999, 65: 5409~5420.
26. Gabor E M, Vries E J, Janssen D B. Efficient recovery of environmental DNA for expression cloning by indirect extraction methods. Microbiol. Ecol. 2003, 44(2): 153~63.
27. Gaisser S, Reather J, Wirtz G, Kellenberger L, Staunton J, Leadlay P F. A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea. Mol Microbiol, 2000, 36(2): 391～401.
28. George K M, Chatterjee D, Gunawardana G, Welty D, Hayman J, Lee R, Small P L. Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence. Science, 1999, 283(5403): 854～857.
29. Gerth K, Bedorf N, Hofle G, Irschik H, Reichenbach H. Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties. J. Antibiot. 1996, 49(6): 560～563.
30. Gillespie D E, Handelsman J, Brady S F, Bettermann A D, Cianciotto N P.. Isolation of antibiotics Turbomycin A and B from a metagenomic library of soil microbial DNA. Appl Environ Microbiol, 2002, 68(9): 4301～4306.
31. Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol. 1998, 5:245-249
32. Handelsman J, Liles M R, Mann D A, Riesenfeld C S, Goodman R M. Cloning the metagenome: culture-independent access to the diversity and functions of theuncultivated microbial world. Functional. Microbial. Genomics. 2003, 6: 241~255.
33. Handlesman J.Sorting out metadenomes. Nat. Biotechnol. 2005, 23:38-39
34. Henne A, Daniel R, Schmitz RA, Gottschalk G. Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate. Appl. Environ. Microbiol. 1999, 65:3901-3907
35. Henne A, Schmitz RA, Bo¨ meke M, Gottschalk G, Daniel R. Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl. Environ. Microbiol. 2000, 66:3113-3116
36. Hertweck C, Xiang L, Kalaitzis J A, Cheng Q, Palzer M, Moore B S. Context-dependent behavior of the enterocin iterative polyketide synthase: a new model for ketoreduction. Chem Biol, 2004, 11 (4): 461～468.
37. Horton T R, Bruns T D. The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Molecular Ecology, 2001, 10(8): 1 855～1 871
38. Holben W E, Jansson J K, Chelm B K, and Tiedje J M. DNA probe method for the detection of specific microorganisms in the soil bacterial community. Applied and Environmental Microbiology. 1988, 54: 703～711.
39. Hurt R A, Qiu X, Wu L, Roh Y, Palumbo A V, Tiedje JM, Zhou J. Simultaneous recovery of RNA and DNA from soils andsediments. Appl. Environ. Microbiol. 2001, 67: 4495~4503
40. Ichinose K, Surti C, Taguchi T, Malpartida F, Booker-Milburn K I, Stephenson G R, Ebizuka Y, Hopwood D A. Proof that the ACTVI genetic region of Streptomyces coelicolor A3 (2) is involved in stereospecific pyran ring formation in the biosynthesis of actinorhodin. Bioorg Med Chem Lett, 1999, 9(3): 395～400.
41. Ichinose K, Ozawa M, Itou K, Kunieda K, Ebizuka Y. Cloning, sequencing and heterologous expression of the medermycin biosynthetic gene cluster of Streptomyces sp. AM-7161: towards comparative analysis of the benzoisochromanequinone gene clusters. Microbiology, 2003, 149(7): 1633～1645.
42. Jacobsen C.S., Rasmussen O.F. Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin, Appl. Environ. Microbiol. 1992, 58: 2458~2462.
43. Janssen P H, Yates P S, Grinton B E, Taylor P M. Sait M. Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl. Environ. Microbiol. 2002, 68: 2391~2396
44. Joseph SJ, Hugenholtz P, Sangwan P, Osborne CA, Janssen PH. Laboratory cultivation of widespread and previously uncultured soil bacteria. Appl. Environ. Microbiol. 2003, 69:7210-7215
45. Kao C M, Katz L, Khosla C. Engineered biosynthesis of a complete macrolactone in a heterologous host. Science, 1994, 265(5171): 509～512.
46. Kaeberlein T, Lewis K, Epstein S. Isolating ‘uncultivable’ microorganisms in a simulated natural environment. Science. 2002, 296:1127-1129
47. Kealey J, Liu L, Santi D V, Betlach M, Barr P J. Productionof a polyketide natural product in non-polyketide producing prokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA, 1998, 95(2): 505～509.
48. Kennedy J, Auclair K, Kendrew S G, Park C, Vederas J C, Hutchinson C R. Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science, 1999, 284(5418): 1368～1372.
49. Kellenberger E. Exploring the unknown: the silent revolution of microbiology. EMBO Rep. 2001, 2:5-7
50. Klappenbach J A, Dunbar J M, Schmidt T M. rRNA operon copy number reflects ecological strategies of bacteria. Appl. Environ. Microbiol. 2000, 66:1328–1333
51. Knietsch A, Waschkowitz T, Bowien S, Henne A, Daniel R. Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli.. Mol. Microbiol. Biotechnol. 2003a, 5: 46~56.
52. Knietsch A, Waschkowitz T, Bowien S, Henne A, Daniel R. Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli. Appl. Environ. Microbiol. 2003b, 69:1408-1416
53. Knietsch A, Bowien S, Whited G, Gottschalk G, Daniel R. Identification andcharacterization of genes encoding coenzyme B12-dependent glycerol and diol dehydratases from metagenomic DNA libraries derived from enrichment cultures. Appl Environ Microbiol, 2003c, 69(6) : 3048～3060.
54. Krsek M, Wellington E M H. Comparisn of different methods for the isoltion and purification of total community DNA from soil [J ]. Journal of Microbiological Methods, 1999, 39(1): 1 - 16.
55. Kuske C R, Banton K, Adorada D L, Stark P C, Hill K K, Jackson P J. Small-Scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Appl. Environ. Microbiol. 1998, 64(7):2463-247.
56. Kwon H J, Smith W C, Xiang L, Shen B. Cloning and heterologous expression of the macrotetrolide biosynthetic gene cluster revealed a novel polyketide synthase that lacks an acyl carrier protein. J Am Chem Soc, 2001, 123(14): 3385～3386.
57. Kwon H J, Smith W C, Scharon A J. C-O bond formation by polyketide synthases. Science, 2002, 297(5585): 1327～1330.
58. Lee S. Y., J. Bollinger, D. Bezdicek, and A. Ogram. Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method. Appl Environ Microbiol. 1996, 62: 3787~3793.
59. Lindahl V., Bakken L.R. Evaluation of methods for extraction of bacterial from soil, FEMS Microbiol. Ecol. 1995, 16: 135~142.
60. Liles M R, Manske B F, Bintrim S B, Handelsman J, Goodman R M. A census of rRNA genes and linked genomic sequenceswithin a soil metagenomic library. Appl. Environ. Microbiol. 2003, 69: 2684~2691.
61. Lloyd-Jones G, Hunter D W F. Comparison of rapid DNA extraction methods applied to contrasting New Zealand soils. Soil. Biol. Biochem. 2001, 33: 2053~2059.
62. Maarit Niemi R., Heiskanen I., Wallenius K., Lindstrom K. Extraction and purification of DNA in hizosphere soil samples for PCR-DGGE analysis of bacterial consortia, J. Microbiol. Methods. 2001, 45: 155~165.
63. MacNeil IA, Tiong CI, Minor C, August PR, Grossmann TH, Lolacono KA, Lynch BA, Phillips T, Narula S, Sundaramoorthi R. Expression and isolation of antimicrobial small molecules from soil DNA libraries. J. Mol. Microbiol.Biotechnol. 2001, 3:301–308
64. Marti T, Hu Z, Pohl N L, Shah A N, Khosla C. Cloning, nucleotide sequence, and heterologous expression of the biosynthetic gene cluster for R1128, a non-steroidal estrogen receptor antagonist. Insights into an unusual priming mechanism. J Biol Chem, 2000, 275(43): 33443～33448.
65. Martin-Laurent F., Philippot L., Hallet S., Chaussod R., Germon J.C., Soulas G., Catroux G. DNA extraction from soils: old bias for new microbial diversity analysis methods, Appl. Environ. Microbiol. 2001, 67: 2354~2359.
66. McDaniel R, Ebert-Khosla S, Hopwood D A, Khosla C. Engineered biosynthesis of novel polyketides. Science, 1993, 262(5139): 1546～1550.
67. Miller D.N., Bryant J.E., Madsen E.L., Ghiorse W.C.. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples, Appl Environ.Microbiol. 1999, 65: 4715~4724.
68. More M L, Herrick J B, Silva M C, et al. Quantitative cell lysis of indigenous microbial DNA from sediment. Appl Environ Microbiol. 1994, 60(5): 1572~1580.
69. Mulert U, Luzhetskyy A, Hofmann C, Mayer A, Bechthold A. Expression of the landomycin biosynthetic gene cluster in a PKS mutant of Streptomyces fradiae is dependent on the coexpression of a putative transcriptional activator gene. FEMS Microbiol Lett, 2004, 230(1): 91～97.
70. Mumy K.L., Findlay R.H. Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR. J Microbiol Methods. 2004, 57: 259~68.
71. Ogram A., Sayler G S, Barkay T. The extraction and purification of microbial DNA from sediment. J Microbio Methods. 1987, 7: 57~66.
72. Patrick Robe, Renaud Nalin, Carmela Capellano, Timothy M. Vogel, Pascal Simonet. Extraction of DNA from soil. European Journal of Soil Biology, 2003 (39): 183～190.
73. Pfelfer B A, Khosla C. Biosynthesis of polyketides in heterologous hosts. Microbiol Mol Biol Rev, 2001, 65 (1): 106～118.
74. Pfeifer B A, Wang C C, Walsh C T, Khosla C. Biosynthesis of Yersiniabactin a complex polyketide-nonribosomal peptide using Escherichia coli as a heterologoushost. Appl Environ Microbiol, 2003, 69(11): 6698～6702.
75. Piel J. A. ,Proc. Natl. Acad. Sci. polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles USA.a long-term fertilizer field experiment. Appl. Environ. Microbiol. 2002,67: 4215- 4224.
76. Pillai S.D., Josephson K.L., Bailey R.L., Gerba C.P., Pepper I.L. Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences, Appl.Environ. Microbiol. 1991, 57 : 2283~2286
77. Proteous LA, Armstrong JL, Seidler RJ, Watrud LS. An effective method to extract DNA from environmental sample for polymerase chain reaction amplification and DNA fingerprint analysis. Curr. Microbiol. 1994, 29: 301-307.
78. Quaiser A, Ochsenreiter T, Klenk H P, Kletzin A, Treusch A H, Meurer G, Eck J, Sensen C W, Schleper C. First insights into the genome of an uncultivated Crenarchaeota from soil. Environ. Microbiol. 2002, 4:603-611
79. Quaiser A, Ochsenreiter T, Lanz C, Schuster S C, Treusch A H, Eck J, Schleper C. Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics. Mol. Microbiol. 2003, 50:563-575
80. Rangaswamy V, Jiralerspong S, Parry R, Bender CL. 1998. Biosynthesis of the Pseudomonas polyketide coronafacic acid requires monofunctional and multifunctional polyketide synthase proteins. Proc Natl Acad Sci USA, 1998, 95(26): 15469～15474.
81. Richardson T H, Tan X, Frey G, Callen W, Cabell M, Lam D, Macomber J, Short J M, Robertson D E, Miller C. A Novel, high Performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable a-amylase. J. Biol. Chem. 2002, 277:26501-26507.
82. Robe P, Nalin R, Capellano C, Vogel T M, Simonet P. Extraction of DNA from soil. Eur J Soil Biol, 2003 39(4): 183～190.
83. Rondon M R, Goodman R M, Handelsman J. The earth’s bounty: assessing and accessing soil microbial diversity. Trends. Biotechnol. 1999, 17:403-409
84. Rondon M R, August P R, Bettermann A D, Brady S F, Gross-man T H, Liles M R, Loiacono K A, Lynch B A, MacNeil I A, Minor C, Tiong C L, Gilman M, OsburneM S, Clardy J, Handelsman J, Goodman R M. Cloning the soil metagenome: a strategy for access-ing the genetic and functional diversity of uncultured microorganisms. Appl Environ Microbiol, 2000, 66(6): 2541～2547.
85. Rosello -Mora R, Amann R: The species concept for prokaryotes. FEMS Microbiol Rev, 2001, 25 (1): 39～67.
86. Roose-Amsaleg C.L., Garnier-Sillam E., Harry M. Extraction and purification of microbial DNA from soil and sediment samples. Applied Soil Ecology. 2001, 18: 47~60.
87. Rose P., Harkin J.M., Hickey W.J. Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction. J Microbiol Methods, 2003, 52: 29~38.
88. Santosa D A. Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies. Mol Biotechnol, 2001, 17(1): 59～64.
89. Schmidt, Delong E, Pace N. Analysis of a marine picoplankton community by 16s rRNA gene cloning and sequencing. Chem. Biol. 1998. 5: 245-249.
90. Shah S, Xue Q, Tang L, Carney J R, Betlach M, McDaniel R. Cloning characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin. J Antibio, 2000, 53(5): 502～508.
91. Shen B. Polyketide biosynthesis beyond the type Ⅰ , Ⅱ and Ⅲ polyketide synthase paradigms. Curr Opion Chem Biol, 2003, 7: 285～295.
92. Sherman D H, Malpartida F, Bibb M J, Kieser H M, Bibb M J, Hopwood D A. Structure and deduced function of the granaticinproducing polyketide synthase gene cluster of Streptomyces. EMBO J, 1989, 8(9): 2717～2725.
93. Sophie Courtois, Carmela M. Cappellano, Maria Ball, Francois-Xavier Francou, Philippe Normand, Gerard Helynck, Asuncion Martinez, Steven J. Kolvek, Joern Hopke, Marcia S. Osburne, Paul R. August, Renaud Nalin. Recombinant Environmental Libraries Provide Access to Microbial Diversity for Drug Discovery from Natural Products. Applied and Environmental Microbiology, 2003, 69(1):49-55.
94. Somerville C C, Knight I T, Straube WL, et al. Simple, rapid method for direct isolation of nucleic acids from aquatic environments. Appl Environ Microbio, 1989, 55 (3) :548 ～554.
95. Steffan R.J., Goksoyr J., Bej A.K., Atlas R.M. Recovery of DNA from soils and sediments, Appl. Environ. Microbiol. 1988, 54: 2908~2915.
96. Stein J L, Marsh T L, Wu K Y, Shizuya H, DeLong E F. Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment front a planktonic marine archaeon. J. Bacteriol. 1996, 178: 591~599.
97. Stokes H W, Holmes A J, Nield B S, Holley M P, Nevalainen K M H, Mabbutt B C, Gillings M R. Gene cassette PCR: sequenceindependent recovery of entire genes from environmental DNA. Appl. Environ. Microbiol. 2001, 67: 5240~5246.
98. Tang L, Fu H, Betlach M C, McDaniel R. Elucidating the mechanism of chain termination switching in the picromycin/ methymycin polyketide synthase. Chem Biol, 1999, 6(8): 553～558.
99. Tang L, Shah S, Chung L, Carney J, Katz L, Khosla C, Julien B. Cloning and heterologous expression of the epothilone gene cluster. Science, 2000, 287(5453): 640～642.
100.Tebbe C. C., Vahjen W. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl Environ Microbiol. 1993, 59: 2657~2665.
101.Tien C.C., Chao C.C. Methods for DNA extraction from various soils: a comparison, J. Appl. Microbiol. 1999, 86: 937~943.
102.Torsvl K, Ovreas VL. Microbial diversity and function in soil. from genes to ecosystems. Curr. Opin. Microbial. 2002, 5:240-245.
103.Torsvik V, Daa FL, Sandaa R-A, Qvreas L. Microbial diversity and function in soil: from genes to ecosystems. Curr. Opin. Microbiol. 2002, 5:240-245
104.Torsvik V, Daa FL, Sandaa R-A, Qvreas L. Novel techniques for analysing microbial diversity in natural and perturbed environments. J. Biotechnol. 1998, 64:53-62
105.Tyson GW, Chapman J, Hugenholtz P, Allen EE, Ram RJ. Community structure andmetabolism through reconstruction of microbial genomes from the environment. Nature. 2004, 428:37-43
106.Uchiyama J, Abe T, Ikemura T, Watanabe K. Substrate-induced gene expression screening of enviromental metagenome libraries for isolation of catabolic genes. Nat. Biotechnol. 2005, 23:88-93
107.Volchegursky Y, Hu Z, Katz L, McDaniel R. Biosynthesis of the anti-parasitic agent megalomicin: transformation of erythromycin to megalomicin in Saccharopolyspora erythraea. Mol Microbiol, 2000, 37(4): 752～762.
108.Wang G Y, Graziani E, Waters B, Pan W, Li X, McDermott J, Meurer G, Saxena G, Andersen R J, Davies J. Novel natural products from soil DNA libraries in a streptomycete host. Org. Lett. 2000, 2(16): 2401~2404.
109.Watanabe K, Rude M A, Walsh C T, Khosla C. Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli. Proc Natl Acad Sci USA, 2003, 100(17): 9774～9778.
110.Weissman K J. Polyketide biosynthesis: understanding and exploiting modularity. Philos Transact A Math Phys Eng Sci, 2004, 362 (1825): 2671～2690.
111.Wery N, Gerike U, Sharman A, Chaudhuri J B, Hough D W, Danson M J. Use of a packed-column bioreactor for isolation of diverse protease-producing bacteria from antarctic soil. Appl. Environ. Microbiol. 2003, 69: 1457~1464
112.Yeates C, Gillings M R, Davison A D, Altavilla N, Veal D A. Methods for microbial DNA extraction from soil for PCR amplification. Biol Proced Online, 1998, 14(1): 40～47.
113.Young C C, Burghoff R L, and Keim L G. Polyvinylpyrrolidoneagrose gel electrophoresis purification of polymerase chain reactionamplifiable DNA from soils. Appl Environ Microbiol 1993 , 59 :1972~1974.
114.Zhou J., Bruns M.A., Tiedje J.M.. DNA recovery from soils of diverse composition, Appl. Environ. Microbiol. 1996, 62 : 316~322.
115.Zhou J, Davey M E, Ficueras J B, et al. Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA. Microbiology. 1997, 143: 3913~3919.
2. 林鹏 中国红树林研究进展 厦门大学学报(自然科学版) 2001, 40(2): 592～603.
3. 腾 应, 骆永明, 赵祥伟, 李振高, 宋 静, 吴龙华. 重金属复合污染农田土壤DNA 的快速提取及其 PCR- DGGE 分析. 土壤学报, 2004, 41 (3): 343～348.
4. 阎 冰, 洪 葵, 许 云, 马 超. 宏基因组克隆——微生物活性物质筛选的新途径. 微生物学通报, 2005, 32(1): 113～117.
5. 张瑞福,曹慧,崔中利,李顺鹏,樊奔. 土壤微生物总 DNA 的提取和纯化. 微生物学报,2003,43(2):276～282.
6. Andrew E. Berry, Claudia Chiocchini, Tina Selby, Margherita Sosio, Elizabeth M.H Wellington. Isolation of high molecular weight DNA from soil for cloning into BAC vectors. FEMS Microbiology Letters. 2003 ,223: 15～20.
7. Amann R I, Ludwig W, Schleifer K H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev, 1995, 59 (1): 143～169.
8. Arslanian R L, Tang L, Blough S, Ma W, Qiu R G, Katz L, Carney J R. A new cytotoxic epothilone from modified polyketide synthases heterologously expressed in Myxococcus xanthus. J Nat Products, 2002, 65(7): 1061～1064.
9. Bakken L.R. Separation and purification of bacteria from soil. Appl. Environ. Microbiol. 1985, 49: 1482~1487.
10. Bakken L.R., Lindahl V Nucleic Acids in the Environment: Methods and Applications, pringer-Verlag, Heidelberg. Germany, 1995: 9~27.
11. Bedford D J, Schweizer E, Hopwood D A, Khosla C. Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor. J Bacteriol, 1995, 177(15): 4544～4548.
12. Berry A E, Chiocchini C, Selby T, Sosio M, Wellington E M. Isolation of high molecular weight DNA from soil for cloning into BAC vectors. FEMS Microbiol Lett, 2003, 223(1): 15～20.
13. Bertrand H, Poly F, Van VT, Lombard N, Nalin R, Vogel TM, Simonet P. High molecular weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction. J Microbiol Meth, 2005, 62(1): 1～11.
14. Burgmann H., Pesaro M., Widmer F., Zeyer J. A strategy for optimizing quality and quantity of DNA extracted from soil, J. Microbiol. Methods, 2001, 45: 7~20.
15. Chandler D.P., Frederickson J.K., Brockman J. Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries. Mol. Ecol. 1997, 6: 475~482.
16. Courtois S., Frostegard A., Goransson P., Depret G., Jeannin P., Simonet P. Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation, Environ.Microbiol. 2001, 3: 431~439.
17. Courtois S, Cappellano CM, Ball M, Francou F-X, Normand P, Helynck G, Martinez A, Kolvek SJ, Hopke J, Osburne MS. Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products. Appl. Environ. Microbiol. 2003, 69: 49–55.
18. Cullen D. W., Hirsch P. R. Simple and rapid method for direct extraction of microbial DNA from soil for PCR. Soil Biol.Biotech, 1998, 30: 983～993.
19. Dojka MA, Harris JK, Pace NR. Expanding the known diversity and environmental distribution of an unculture phylogenetic division of bacteria. Environ. Microbiol. 2000, 66: 1617-1621.
20. Entcheva P, Liebl W, Johann A, HartschT, Streit WR. Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia. Appl. Environ.Microbiol. 2001, 67:89–99
21. Elsas J D, Duarte G F, Rosado A S. Microbiological and molecular biological methods for monitoring microbial inoculants and their effects in the soil environment. Jour Microbial Methods. 1998, 32 : 133～154.
22. Faegri A., Torsvik V.L., Goksoyr J. Bacterial and fungal activities in soil: separation of bacteria and fungi by a rapid fractionated centrifugation technique, Soil Biol. Biochem. 1977, 9: 105~112.
23. Fernandez-Moreno M A, Martinez E, Boto L, Hopwood DA, Malpartida F. Nucleotide sequence and deduced functions of a set of cotranscribed genes of S t reptomyces coelicolor A 3 (2) including the polyketide synthase for the antibiotic actinorhodin. J Biol Chem, 1992, 267(27):19278～19290.
24. Fu H, Ebert-Khosla S, Hopwood D A, Khosla C. Relaxed specificity of the oxytetracycline polyketide synthase for an acetate primer in the absence of a malonyamyl primer. J Am Chem Soc, 1994, 116(14): 6443～6444.
25. Frostegard A, Courtois S, Ramisse V, et al. Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbio.1999, 65: 5409~5420.
26. Gabor E M, Vries E J, Janssen D B. Efficient recovery of environmental DNA for expression cloning by indirect extraction methods. Microbiol. Ecol. 2003, 44(2): 153~63.
27. Gaisser S, Reather J, Wirtz G, Kellenberger L, Staunton J, Leadlay P F. A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea. Mol Microbiol, 2000, 36(2): 391～401.
28. George K M, Chatterjee D, Gunawardana G, Welty D, Hayman J, Lee R, Small P L. Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence. Science, 1999, 283(5403): 854～857.
29. Gerth K, Bedorf N, Hofle G, Irschik H, Reichenbach H. Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties. J. Antibiot. 1996, 49(6): 560～563.
30. Gillespie D E, Handelsman J, Brady S F, Bettermann A D, Cianciotto N P.. Isolation of antibiotics Turbomycin A and B from a metagenomic library of soil microbial DNA. Appl Environ Microbiol, 2002, 68(9): 4301～4306.
31. Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol. 1998, 5:245-249
32. Handelsman J, Liles M R, Mann D A, Riesenfeld C S, Goodman R M. Cloning the metagenome: culture-independent access to the diversity and functions of theuncultivated microbial world. Functional. Microbial. Genomics. 2003, 6: 241~255.
33. Handlesman J.Sorting out metadenomes. Nat. Biotechnol. 2005, 23:38-39
34. Henne A, Daniel R, Schmitz RA, Gottschalk G. Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate. Appl. Environ. Microbiol. 1999, 65:3901-3907
35. Henne A, Schmitz RA, Bo¨ meke M, Gottschalk G, Daniel R. Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl. Environ. Microbiol. 2000, 66:3113-3116
36. Hertweck C, Xiang L, Kalaitzis J A, Cheng Q, Palzer M, Moore B S. Context-dependent behavior of the enterocin iterative polyketide synthase: a new model for ketoreduction. Chem Biol, 2004, 11 (4): 461～468.
37. Horton T R, Bruns T D. The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Molecular Ecology, 2001, 10(8): 1 855～1 871
38. Holben W E, Jansson J K, Chelm B K, and Tiedje J M. DNA probe method for the detection of specific microorganisms in the soil bacterial community. Applied and Environmental Microbiology. 1988, 54: 703～711.
39. Hurt R A, Qiu X, Wu L, Roh Y, Palumbo A V, Tiedje JM, Zhou J. Simultaneous recovery of RNA and DNA from soils andsediments. Appl. Environ. Microbiol. 2001, 67: 4495~4503
40. Ichinose K, Surti C, Taguchi T, Malpartida F, Booker-Milburn K I, Stephenson G R, Ebizuka Y, Hopwood D A. Proof that the ACTVI genetic region of Streptomyces coelicolor A3 (2) is involved in stereospecific pyran ring formation in the biosynthesis of actinorhodin. Bioorg Med Chem Lett, 1999, 9(3): 395～400.
41. Ichinose K, Ozawa M, Itou K, Kunieda K, Ebizuka Y. Cloning, sequencing and heterologous expression of the medermycin biosynthetic gene cluster of Streptomyces sp. AM-7161: towards comparative analysis of the benzoisochromanequinone gene clusters. Microbiology, 2003, 149(7): 1633～1645.
42. Jacobsen C.S., Rasmussen O.F. Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin, Appl. Environ. Microbiol. 1992, 58: 2458~2462.
43. Janssen P H, Yates P S, Grinton B E, Taylor P M. Sait M. Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl. Environ. Microbiol. 2002, 68: 2391~2396
44. Joseph SJ, Hugenholtz P, Sangwan P, Osborne CA, Janssen PH. Laboratory cultivation of widespread and previously uncultured soil bacteria. Appl. Environ. Microbiol. 2003, 69:7210-7215
45. Kao C M, Katz L, Khosla C. Engineered biosynthesis of a complete macrolactone in a heterologous host. Science, 1994, 265(5171): 509～512.
46. Kaeberlein T, Lewis K, Epstein S. Isolating ‘uncultivable’ microorganisms in a simulated natural environment. Science. 2002, 296:1127-1129
47. Kealey J, Liu L, Santi D V, Betlach M, Barr P J. Productionof a polyketide natural product in non-polyketide producing prokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA, 1998, 95(2): 505～509.
48. Kennedy J, Auclair K, Kendrew S G, Park C, Vederas J C, Hutchinson C R. Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science, 1999, 284(5418): 1368～1372.
49. Kellenberger E. Exploring the unknown: the silent revolution of microbiology. EMBO Rep. 2001, 2:5-7
50. Klappenbach J A, Dunbar J M, Schmidt T M. rRNA operon copy number reflects ecological strategies of bacteria. Appl. Environ. Microbiol. 2000, 66:1328–1333
51. Knietsch A, Waschkowitz T, Bowien S, Henne A, Daniel R. Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli.. Mol. Microbiol. Biotechnol. 2003a, 5: 46~56.
52. Knietsch A, Waschkowitz T, Bowien S, Henne A, Daniel R. Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli. Appl. Environ. Microbiol. 2003b, 69:1408-1416
53. Knietsch A, Bowien S, Whited G, Gottschalk G, Daniel R. Identification andcharacterization of genes encoding coenzyme B12-dependent glycerol and diol dehydratases from metagenomic DNA libraries derived from enrichment cultures. Appl Environ Microbiol, 2003c, 69(6) : 3048～3060.
54. Krsek M, Wellington E M H. Comparisn of different methods for the isoltion and purification of total community DNA from soil [J ]. Journal of Microbiological Methods, 1999, 39(1): 1 - 16.
55. Kuske C R, Banton K, Adorada D L, Stark P C, Hill K K, Jackson P J. Small-Scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Appl. Environ. Microbiol. 1998, 64(7):2463-247.
56. Kwon H J, Smith W C, Xiang L, Shen B. Cloning and heterologous expression of the macrotetrolide biosynthetic gene cluster revealed a novel polyketide synthase that lacks an acyl carrier protein. J Am Chem Soc, 2001, 123(14): 3385～3386.
57. Kwon H J, Smith W C, Scharon A J. C-O bond formation by polyketide synthases. Science, 2002, 297(5585): 1327～1330.
58. Lee S. Y., J. Bollinger, D. Bezdicek, and A. Ogram. Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method. Appl Environ Microbiol. 1996, 62: 3787~3793.
59. Lindahl V., Bakken L.R. Evaluation of methods for extraction of bacterial from soil, FEMS Microbiol. Ecol. 1995, 16: 135~142.
60. Liles M R, Manske B F, Bintrim S B, Handelsman J, Goodman R M. A census of rRNA genes and linked genomic sequenceswithin a soil metagenomic library. Appl. Environ. Microbiol. 2003, 69: 2684~2691.
61. Lloyd-Jones G, Hunter D W F. Comparison of rapid DNA extraction methods applied to contrasting New Zealand soils. Soil. Biol. Biochem. 2001, 33: 2053~2059.
62. Maarit Niemi R., Heiskanen I., Wallenius K., Lindstrom K. Extraction and purification of DNA in hizosphere soil samples for PCR-DGGE analysis of bacterial consortia, J. Microbiol. Methods. 2001, 45: 155~165.
63. MacNeil IA, Tiong CI, Minor C, August PR, Grossmann TH, Lolacono KA, Lynch BA, Phillips T, Narula S, Sundaramoorthi R. Expression and isolation of antimicrobial small molecules from soil DNA libraries. J. Mol. Microbiol.Biotechnol. 2001, 3:301–308
64. Marti T, Hu Z, Pohl N L, Shah A N, Khosla C. Cloning, nucleotide sequence, and heterologous expression of the biosynthetic gene cluster for R1128, a non-steroidal estrogen receptor antagonist. Insights into an unusual priming mechanism. J Biol Chem, 2000, 275(43): 33443～33448.
65. Martin-Laurent F., Philippot L., Hallet S., Chaussod R., Germon J.C., Soulas G., Catroux G. DNA extraction from soils: old bias for new microbial diversity analysis methods, Appl. Environ. Microbiol. 2001, 67: 2354~2359.
66. McDaniel R, Ebert-Khosla S, Hopwood D A, Khosla C. Engineered biosynthesis of novel polyketides. Science, 1993, 262(5139): 1546～1550.
67. Miller D.N., Bryant J.E., Madsen E.L., Ghiorse W.C.. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples, Appl Environ.Microbiol. 1999, 65: 4715~4724.
68. More M L, Herrick J B, Silva M C, et al. Quantitative cell lysis of indigenous microbial DNA from sediment. Appl Environ Microbiol. 1994, 60(5): 1572~1580.
69. Mulert U, Luzhetskyy A, Hofmann C, Mayer A, Bechthold A. Expression of the landomycin biosynthetic gene cluster in a PKS mutant of Streptomyces fradiae is dependent on the coexpression of a putative transcriptional activator gene. FEMS Microbiol Lett, 2004, 230(1): 91～97.
70. Mumy K.L., Findlay R.H. Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR. J Microbiol Methods. 2004, 57: 259~68.
71. Ogram A., Sayler G S, Barkay T. The extraction and purification of microbial DNA from sediment. J Microbio Methods. 1987, 7: 57~66.
72. Patrick Robe, Renaud Nalin, Carmela Capellano, Timothy M. Vogel, Pascal Simonet. Extraction of DNA from soil. European Journal of Soil Biology, 2003 (39): 183～190.
73. Pfelfer B A, Khosla C. Biosynthesis of polyketides in heterologous hosts. Microbiol Mol Biol Rev, 2001, 65 (1): 106～118.
74. Pfeifer B A, Wang C C, Walsh C T, Khosla C. Biosynthesis of Yersiniabactin a complex polyketide-nonribosomal peptide using Escherichia coli as a heterologoushost. Appl Environ Microbiol, 2003, 69(11): 6698～6702.
75. Piel J. A. ,Proc. Natl. Acad. Sci. polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles USA.a long-term fertilizer field experiment. Appl. Environ. Microbiol. 2002,67: 4215- 4224.
76. Pillai S.D., Josephson K.L., Bailey R.L., Gerba C.P., Pepper I.L. Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences, Appl.Environ. Microbiol. 1991, 57 : 2283~2286
77. Proteous LA, Armstrong JL, Seidler RJ, Watrud LS. An effective method to extract DNA from environmental sample for polymerase chain reaction amplification and DNA fingerprint analysis. Curr. Microbiol. 1994, 29: 301-307.
78. Quaiser A, Ochsenreiter T, Klenk H P, Kletzin A, Treusch A H, Meurer G, Eck J, Sensen C W, Schleper C. First insights into the genome of an uncultivated Crenarchaeota from soil. Environ. Microbiol. 2002, 4:603-611
79. Quaiser A, Ochsenreiter T, Lanz C, Schuster S C, Treusch A H, Eck J, Schleper C. Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics. Mol. Microbiol. 2003, 50:563-575
80. Rangaswamy V, Jiralerspong S, Parry R, Bender CL. 1998. Biosynthesis of the Pseudomonas polyketide coronafacic acid requires monofunctional and multifunctional polyketide synthase proteins. Proc Natl Acad Sci USA, 1998, 95(26): 15469～15474.
81. Richardson T H, Tan X, Frey G, Callen W, Cabell M, Lam D, Macomber J, Short J M, Robertson D E, Miller C. A Novel, high Performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable a-amylase. J. Biol. Chem. 2002, 277:26501-26507.
82. Robe P, Nalin R, Capellano C, Vogel T M, Simonet P. Extraction of DNA from soil. Eur J Soil Biol, 2003 39(4): 183～190.
83. Rondon M R, Goodman R M, Handelsman J. The earth’s bounty: assessing and accessing soil microbial diversity. Trends. Biotechnol. 1999, 17:403-409
84. Rondon M R, August P R, Bettermann A D, Brady S F, Gross-man T H, Liles M R, Loiacono K A, Lynch B A, MacNeil I A, Minor C, Tiong C L, Gilman M, OsburneM S, Clardy J, Handelsman J, Goodman R M. Cloning the soil metagenome: a strategy for access-ing the genetic and functional diversity of uncultured microorganisms. Appl Environ Microbiol, 2000, 66(6): 2541～2547.
85. Rosello -Mora R, Amann R: The species concept for prokaryotes. FEMS Microbiol Rev, 2001, 25 (1): 39～67.
86. Roose-Amsaleg C.L., Garnier-Sillam E., Harry M. Extraction and purification of microbial DNA from soil and sediment samples. Applied Soil Ecology. 2001, 18: 47~60.
87. Rose P., Harkin J.M., Hickey W.J. Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction. J Microbiol Methods, 2003, 52: 29~38.
88. Santosa D A. Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies. Mol Biotechnol, 2001, 17(1): 59～64.
89. Schmidt, Delong E, Pace N. Analysis of a marine picoplankton community by 16s rRNA gene cloning and sequencing. Chem. Biol. 1998. 5: 245-249.
90. Shah S, Xue Q, Tang L, Carney J R, Betlach M, McDaniel R. Cloning characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin. J Antibio, 2000, 53(5): 502～508.
91. Shen B. Polyketide biosynthesis beyond the type Ⅰ , Ⅱ and Ⅲ polyketide synthase paradigms. Curr Opion Chem Biol, 2003, 7: 285～295.
92. Sherman D H, Malpartida F, Bibb M J, Kieser H M, Bibb M J, Hopwood D A. Structure and deduced function of the granaticinproducing polyketide synthase gene cluster of Streptomyces. EMBO J, 1989, 8(9): 2717～2725.
93. Sophie Courtois, Carmela M. Cappellano, Maria Ball, Francois-Xavier Francou, Philippe Normand, Gerard Helynck, Asuncion Martinez, Steven J. Kolvek, Joern Hopke, Marcia S. Osburne, Paul R. August, Renaud Nalin. Recombinant Environmental Libraries Provide Access to Microbial Diversity for Drug Discovery from Natural Products. Applied and Environmental Microbiology, 2003, 69(1):49-55.
94. Somerville C C, Knight I T, Straube WL, et al. Simple, rapid method for direct isolation of nucleic acids from aquatic environments. Appl Environ Microbio, 1989, 55 (3) :548 ～554.
95. Steffan R.J., Goksoyr J., Bej A.K., Atlas R.M. Recovery of DNA from soils and sediments, Appl. Environ. Microbiol. 1988, 54: 2908~2915.
96. Stein J L, Marsh T L, Wu K Y, Shizuya H, DeLong E F. Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment front a planktonic marine archaeon. J. Bacteriol. 1996, 178: 591~599.
97. Stokes H W, Holmes A J, Nield B S, Holley M P, Nevalainen K M H, Mabbutt B C, Gillings M R. Gene cassette PCR: sequenceindependent recovery of entire genes from environmental DNA. Appl. Environ. Microbiol. 2001, 67: 5240~5246.
98. Tang L, Fu H, Betlach M C, McDaniel R. Elucidating the mechanism of chain termination switching in the picromycin/ methymycin polyketide synthase. Chem Biol, 1999, 6(8): 553～558.
99. Tang L, Shah S, Chung L, Carney J, Katz L, Khosla C, Julien B. Cloning and heterologous expression of the epothilone gene cluster. Science, 2000, 287(5453): 640～642.
100.Tebbe C. C., Vahjen W. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl Environ Microbiol. 1993, 59: 2657~2665.
101.Tien C.C., Chao C.C. Methods for DNA extraction from various soils: a comparison, J. Appl. Microbiol. 1999, 86: 937~943.
102.Torsvl K, Ovreas VL. Microbial diversity and function in soil. from genes to ecosystems. Curr. Opin. Microbial. 2002, 5:240-245.
103.Torsvik V, Daa FL, Sandaa R-A, Qvreas L. Microbial diversity and function in soil: from genes to ecosystems. Curr. Opin. Microbiol. 2002, 5:240-245
104.Torsvik V, Daa FL, Sandaa R-A, Qvreas L. Novel techniques for analysing microbial diversity in natural and perturbed environments. J. Biotechnol. 1998, 64:53-62
105.Tyson GW, Chapman J, Hugenholtz P, Allen EE, Ram RJ. Community structure andmetabolism through reconstruction of microbial genomes from the environment. Nature. 2004, 428:37-43
106.Uchiyama J, Abe T, Ikemura T, Watanabe K. Substrate-induced gene expression screening of enviromental metagenome libraries for isolation of catabolic genes. Nat. Biotechnol. 2005, 23:88-93
107.Volchegursky Y, Hu Z, Katz L, McDaniel R. Biosynthesis of the anti-parasitic agent megalomicin: transformation of erythromycin to megalomicin in Saccharopolyspora erythraea. Mol Microbiol, 2000, 37(4): 752～762.
108.Wang G Y, Graziani E, Waters B, Pan W, Li X, McDermott J, Meurer G, Saxena G, Andersen R J, Davies J. Novel natural products from soil DNA libraries in a streptomycete host. Org. Lett. 2000, 2(16): 2401~2404.
109.Watanabe K, Rude M A, Walsh C T, Khosla C. Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli. Proc Natl Acad Sci USA, 2003, 100(17): 9774～9778.
110.Weissman K J. Polyketide biosynthesis: understanding and exploiting modularity. Philos Transact A Math Phys Eng Sci, 2004, 362 (1825): 2671～2690.
111.Wery N, Gerike U, Sharman A, Chaudhuri J B, Hough D W, Danson M J. Use of a packed-column bioreactor for isolation of diverse protease-producing bacteria from antarctic soil. Appl. Environ. Microbiol. 2003, 69: 1457~1464
112.Yeates C, Gillings M R, Davison A D, Altavilla N, Veal D A. Methods for microbial DNA extraction from soil for PCR amplification. Biol Proced Online, 1998, 14(1): 40～47.
113.Young C C, Burghoff R L, and Keim L G. Polyvinylpyrrolidoneagrose gel electrophoresis purification of polymerase chain reactionamplifiable DNA from soils. Appl Environ Microbiol 1993 , 59 :1972~1974.
114.Zhou J., Bruns M.A., Tiedje J.M.. DNA recovery from soils of diverse composition, Appl. Environ. Microbiol. 1996, 62 : 316~322.
115.Zhou J, Davey M E, Ficueras J B, et al. Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA. Microbiology. 1997, 143: 3913~3919.