Please wait a minute...
Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2011, Vol. 5 Issue (4) : 576-580    https://doi.org/10.1007/s11703-011-1119-x
RESEARCH ARTICLE
Construction of a Fosmid genomic library of Streptomyces roseoflavus Men-myco-93-63
Weiming SUN1,2, Wei GUO1,2,3(), Daqun LIU1(), Tinghui LIU1, Lina FENG1, Lianna LIU2, Yaning LI1, Yakun ZHANG2
1. Biological Control Center of Plant Diseases and Plant Pests of Hebei Province, College of Plant Protection, Agricultural University of Hebei, Baoding 071000, China; 2. College of Life Sciences, Agricultural University of Hebei, Baoding 071000, China; 3. National Engineering Research Center for Agriculture in Northern Mountainous Areas, Baoding 071000, China
 Download: PDF(172 KB)   HTML
 Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract

To clone the antibiotic biosynthesis gene cluster of Streptomyces roseoflavus Men-myco-93-63, we constructed a Fosmid genomic library. The genomic DNA of the strain Men-myco-93-63 was isolated by the modified CTAB procedure, and the size of most genomic DNA fragments was larger than 150 kb. Then, a Fosmid genomic library containing more than 6000 clones was constructed. The average size of the inserted DNA in recombinant plasmids was 38.1 kb, and the probability of harboring any gene in the genome of the strain Men-myco-93-63 was 99.99%. The library coverage was at least a 10-fold genome equivalent. Therefore, the constructed Fosmid library meets the requirements as a standard genomic library

Keywords Streptomyces roseoflavus      Fosmid      genomic library     
Corresponding Author(s): GUO Wei,Email:guowei@hebau.edu.cn; LIU Daqun,Email:ldq@hebau.edu.cn   
Issue Date: 05 December 2011
 Cite this article:   
Weiming SUN,Wei GUO,Daqun LIU, et al. Construction of a Fosmid genomic library of Streptomyces roseoflavus Men-myco-93-63[J]. Front Agric Chin, 2011, 5(4): 576-580.
 URL:  
https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1119-x
https://academic.hep.com.cn/fag/EN/Y2011/V5/I4/576
Fig.1  Detection of genomic DNA. M is MidRangeII PFG markers, and 1 is genomic DNA.
Fig.2  Sheared DNA. M is 42-kb Fosmid Control DNA; M2 is λ-Mix Markers. 1-4 are genomic DNA aspirated and expelled 40, 50, 60, and 70 times, successively.
Fig.3  Size-fractionated DNA. Note: M is 42-kb Fosmid Control DNA. 1-4 are the concentration of λ-Mix markers at 200, 300, 400, and 500 μg/mL.
Fig.4  Detecting the size of the insert DNA. Note: M is λ-Mix markers. M is λ-T14 markers. Other lanes are 19 clones digested with I.
1 Cantor C R, Smith C L, Mathew M K (1988). Pulsed-field gel electrophoresis of very large DNA molecules.Annu Rev Biophys Biophys Chem , 17(1): 287–304
doi: 10.1146/annurev.bb.17.060188.001443 pmid:3293589
2 Di D P (2006). The extraction of the antibiotic and related genes cloning from Streptomyces roseoflavus Men-myco-93-63. Dissertation for the Doctoral Degree, Baoding: Agricultural University of Hebei, 31–46 (in Chinese)
3 Gravius B, Cullum J, Hranueli D (1994). High G + C-content DNA markers for pulsed-field gel electrophoresis.Biotechniques , 16(1): 52
pmid:8136139
4 Hopwood D A, Bibb M J, Chater K F, Kieser T, Bruton C J, Kieser H M, Lydiate D J, Smith C P, Ward J M, Schrempf H (1985). Genetic Manipulation of Streptomyces: A Laboratory Manual. Norwich: the John Innes Foundation , 156–157
5 Hopwood D A, Kieser T (1990). The Streptomyces genome. In: Drlica K, Riley M, eds. The Bacterial Chromosome. Washington DC: ASM Press, 147–162
6 Hradecna Z, Wild J, Szybalski W (1998). Conditionally amplifiable inserts in pBAC vectors.Microb Comp Genom , 3: 58
7 Liu D Q (1992). Biological control of Streptomyces scabies and other plant pathogens. Dissertation for the Doctoral Degree , USA: the University of Minnesota, 8–20
8 Liu L Q (2006). The study on the chitinase gene and the antibiotic biosynthesis gene cluster of Streptomyces roseoflavus. Dissertation for the Doctoral Degree , Baoding: Agricultural University of Hebei, 48–77 (in Chinese)
9 MacNeil T, Gibbons P H (1986). Characterization of the Streptomyces plasmid pVE1.Plasmid , 16(3): 182–194 0.1016/0147-619X(86)90056-9 pmid:3027726
10 Maniloff J (1989). Anomalous values of Mycoplasma genomes sizes determined by pulse-field gel electrophoresis.Nucleic Acids Res , 17(3): 1268
doi: 10.1093/nar/17.3.1268 pmid:2922272
11 Meng Q F, Yang W X, Zhang Q L, Liu D Q (2004). Field evaluation of antagonistic Streptomyces Men-myco-93-63 preparation in biocontrol of cotton Verticillium Wilt. In: Proceedings of the 15th International Plant Protection Congress . Beijing: Foreign Languages Press, 153
12 Murray M G, Thompson W F (1980). Rapid isolation of high molecular weight plant DNA.Nucleic Acids Res , 8(19): 4321–4326
doi: 10.1093/nar/8.19.4321 pmid:7433111
13 Pospiech A, Neumann B (1995). A versatile quick-prep of genomic DNA from gram-positive bacteria.Trends Genet , 11(6): 217–218
doi: 10.1016/S0168-9525(00)89052-6 pmid:7638902
14 Sambrook J, Fritsch E F, Maniatis T (1989). Molecular Cloning: A Laboratory Manual. 2nd ed. New York: CSH Laboratory Press, 457
15 Smith C L, Matsumoto T, Niwa O, Klco S, Fan J B, Yanagida M, Cantor C R (1987). An electrophoretic karyotype for Schizosaccharomyces pombe by pulsed field gel electrophoresis. Nucleic Acids Res , 15(11): 4481–4489
doi: 10.1093/nar/15.11.4481 pmid:3295780
16 Sun W M (2009). Study on the antibiotic biosynthetic gene related linear plasmid of Streptomyces roseoflavus Men-myco-93-63. Dissertation for the Masteral Degree , Baoding: Agricultural University of Hebei, 3–4 (in Chinese)
17 Sun W M, Guo W, Liu D Q (2008). Efficient detection and analysis of linear plasmid in Streptomyces Men-myco-93-63.Acta Microbiol Sin , 48(12): 1671–1674 (in Chinese)
18 Tobias K, Mervyn J B, Mark J B, Keith F C, David A H (2000). Practional Streptomyces Genetics. Norwich: the John Innes Foundation, 170–171
19 Wild J, Hradecna Z, Szybalski W (2001). Single-copy/high-copy (SC/HC) pBAC/oriV novel vectors for genomics and gene expression.Plasmid , 45: 142–143
20 Wild J, Hradecna Z, Szybalski W (2002). Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.Genome Res , 12(9): 1434–1444
doi: 10.1101/gr.130502 pmid:12213781
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed