Vector for knocking out streptomycete gene as well as constructing method and application of same

A Streptomyces and gene technology, applied in the field of Streptomyces gene knockout vectors, can solve the problems of poor colony PCR stability, false positives, inability to improve the screening efficiency of double-crossover transformants, etc., to achieve simple gene knockout and make up for false positives Effect

Active Publication Date: 2013-05-01
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Claims
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Problems solved by technology

However, when antibiotic resistance and sensitivity are used for photocopy screening, there are a large number of false positives; and direct PCR amplification screening will consume a lot of manpower and material resources. The cell wall is thick, and the stability of colony PCR is very poor, so the method of using antibiotic resistance and PCR amplification can not improve the screening efficiency of double crossover transformants

Method used

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  • Vector for knocking out streptomycete gene as well as constructing method and application of same
  • Vector for knocking out streptomycete gene as well as constructing method and application of same
  • Vector for knocking out streptomycete gene as well as constructing method and application of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, construction is used for knocking out the knockout vector of the Streptomyces gene that does not produce glucanase

[0055] 1. Cloning of Bacillus amyloliquefaciens glucanase gene

[0056] Using a Bacillus amyloliquefaciens YW26 genomic DNA screened by our laboratory that has significant inhibitory effects on a variety of plant pathogenic fungi as a template, according to the sequence of the Bacillus amyloliquefaciens endoglucanase gene (DQ782954.1) in GenBank Design primers: P1 (5'-ATGAAACGGTCAATTTCTAT-3') and P2 (5'-CTAATTGGGTTCTGATCC-3') for PCR amplification. The reaction was pre-denatured at 95°C for 5min; 30 cycles (94°C for 40s, 55°C for 30s, 72°C for 1min); 72°C for 10min. The amplified 1500bp fragment was ligated with the plasmid pMD18-T vector to obtain pMD18-glu, which was transformed into Escherichia coli DH5α, and the obtained positive transformants were verified by PCR and enzyme digestion through ampicillin resistance screening. Sequencing...

Embodiment 2

[0062] Example 2. Construction of a knockout vector for knockout of the positive regulatory gene slnM for natamycin synthesis in Streptomyces that does not produce glucanase

[0063] The coding sequence of the natamycin synthesis positive regulatory gene slnM is SEQ ID No.4, which encodes a protein whose amino acid sequence is shown in SEQ ID No.3.

[0064] Firstly, the nucleotide sequence is constructed as a DNA fragment of the knockout Streptomyces slnM of SEQ ID No.5. SEQ ID No.5 consists of 4144 nucleotides, and positions 1-8 are EcoRI recognition sites and protected bases. Positions 29-1221 are the upstream homology arm of the natamycin synthesis positive regulatory gene slnM, positions 1265-2911 are thiostrepton resistance gene (tsr), and positions 2956-4117 are positive regulators of natamycin synthesis In the downstream homology arm of the regulatory gene slnM, positions 4136-4144 are HindIII recognition sites and protective bases.

[0065] The DNA fragment of the kno...

Embodiment 3

[0066] Example 3. Construction of a mutant strain in which the positive regulator gene slnM for natamycin synthesis in streptomyces that does not produce glucanase is knocked out

[0067] 1. The pKCG + m - 1139 vector transformed demethylated E.coliET12567 (pUZ8002)

[0068] pKCG by heat shock method + m - The 1139 vector was introduced into E.coliET12567 (pUZ8002) and was screened for resistance to 15 μg / ml thiostrepton to obtain pKCG + m - 1139 vector recombinant strain E.coliET12567(pUZ8002) / pKCG + m - 1139.

[0069] 2. Preparation of slnM knockout strain

[0070] Reference (Bierman M et al.1992) for amphiphilic conjugation pKCG + m - 1139 was introduced into Streptomyces lydidii, and the specific method was as follows:

[0071] The recombinant strain E.coliET12567(pUZ8002) / pKCG + m - 1139 was inoculated into LB liquid medium containing chloramphenicol, kanamycin and apramycin resistance, cultured overnight at 37°C, 200rpm, and transferred to fresh LB medium at...

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Abstract

The invention discloses a vector for knocking out a streptomycete gene as well as a constructing method and an application of the vector. A recombinant vector is a recombinant DNA (Deoxyribose Nucleic Acid) obtained by inserting a locus ClaI of pKC1139 into a glucanase gene. In actual application, a streptomycete target gene knockout vector can be obtained after a DNA fragment for knocking out a streptomycete target gene is inserted in a multiple clone site of the recombinant vector; after the streptomycete target gene knockout vector is introduced into streptomycete with the target gene to be knocked out, transformants with thiostrepton resistances are screened; cellulose degradation and screening are carried out on the transformants with the thiostrepton resistances; and specifically, a homologous double-exchange transformant can be obtained through inserting the transformants with the thiostrepton resistances into a cellulose screening flat plate and then carrying out Congo red staining on the transformants with the thiostrepton resistances so as to directly observe degradation situation of the cellulose. Therefore, the phenomenon of incomplete knockout of the gene due to false positive antibiotics resistance can be avoided. Meanwhile, PCR verifications of a large amount of transformants can be avoided. The vector for knocking out the streptomycete gene, disclosed by the invention, can be used for screening any gene knockout double-exchange transformant of strains with no glucanase yield.

Description

technical field [0001] The invention relates to a vector for knocking out Streptomyces gene, its construction method and application. Background technique [0002] Streptomyces is an aerobic Gram-positive soil bacterium with a branched mycelium structure, belonging to the Actinobacteriaceae Streptomycetaceae of the Prokaryote kingdom, and is one of the main microbial groups in soil. Its G%+C% content is as high as 70%-80%, and it is a class of organisms with the highest G%+C% content known in nature so far. Although Streptomyces belongs to prokaryotes, they have a very complex cell differentiation mechanism, which is a good material for studying the regulation mechanism of gene expression in time, space and program. Secondly, Streptomyces has rich secondary metabolic diversity, nearly 70 in nature. % of antibiotics are produced by Streptomyces and its relative actinomycetes. In addition, Streptomyces also produces other useful secondary metabolites such as antineoplastic dr...

Claims

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Application Information

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IPC IPC(8): C12N15/65C12N15/63C12N1/13C12N1/15C12N1/19C12N1/21C12N15/09C12R1/465
Inventor 吴慧玲刘伟成董丹刘霆卢彩鸽张涛涛张殿朋田兆丰
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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