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Glutamine synthetase exogenous expression method based on bacillus megaterium

A technology of Bacillus megaterium and glutamine, applied in the field of genetic engineering, can solve the problems of low nitrogen fertilizer utilization rate and environmental pollution, and achieve the effect of increasing the expression amount

Inactive Publication Date: 2014-02-19
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the environmental pollution caused by the low utilization rate of nitrogen fertilizer and excessive use has become a worldwide problem

Method used

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  • Glutamine synthetase exogenous expression method based on bacillus megaterium
  • Glutamine synthetase exogenous expression method based on bacillus megaterium
  • Glutamine synthetase exogenous expression method based on bacillus megaterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Cultivation of Bacillus megaterium NCT-2

[0032] Bacillus megaterium NCT-2 was isolated from a greenhouse with 12-15 years of facility cultivation in Chongming Island, Shanghai, and the preservation number is CGMCC NO.4698. The bacteria were inoculated in LB liquid medium and cultured at 32°C for OD 600 To about 1.5, centrifuge to collect the bacteria and extract the bacterial genome DNA.

[0033] The components of the LB liquid medium are: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, 1 L of deionized water, and pH 6.8-7.2. LB solid medium is 15-20g agar added on the basis of LB liquid medium.

Embodiment 2

[0035] Sequence Verification of Bacillus megaterium Glutamine Synthetase Gene (Bm-Gln A)

[0036] According to the results of whole genome sequencing, design PCR primers:

[0037] Forward primer Gln A-F: 5'-ATGGCAAAGTACACAAAAGAAGATA-3'

[0038] Reverse primer Gln A-R:5'-TTAATATTGGCTCATGTATTGGTCAC-3'

[0039] Using the genomic DNA of Bacillus megaterium NCT-2 as a template, the above two pairs of primers were used for amplification. The PCR conditions were: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 45 s, annealing at 53°C for 30 s, extension at 72°C for 1 min; and final extension at 72°C for 10 min after 30 cycles.

[0040] The PCR amplification product is carried out electrophoresis detection, and the result shows that the obtained fragment is about 1.3Kb (see figure 1 ); Then the PCR product was gel-cut and recovered and connected to the pET-41a vector and then sent to the company for sequencing. The nucleotide sequence obtained after sequencing is cons...

Embodiment 3

[0042] Construction of recombinant expression vector

[0043] According to the glutamine synthetase sequence (Bm-Gln A) obtained by sequencing, primers were designed to amplify the gene and connected to the pET-41a expression vector, and then poured into Escherichia coli BL21 (DE3) for induced expression.

[0044] Primers were designed as follows:

[0045] Forward primer GlnA-Eco R I-F:

[0046] 5'-CGGAATTCATGGCAAAGTACACAAAAGAAGATA-3'

[0047] Reverse primer Gln A-Stu I-R:

[0048] 5'-AAGGCCTTTAATATTGGCTCATGTATTGGTCAC-3'

[0049] EcoR I and Stu I restriction sites were designed at the 5' ends of the upstream and downstream primers for connection to the expression vector

[0050]For pET-41a, the glutamine synthetase gene was amplified with primers containing restriction sites, and the PCR product Bm-Gln A' was recovered and connected to T. Transfer the ligation product into DH5α Escherichia coli competent cells, pick the positive clones and shake them to recover their plas...

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Abstract

The invention provides a glutamine synthetase exogenous expression method based on bacillus megaterium in the technical field of genetic engineering. Glutamine synthetase gene sequences obtained through sequencing are amplified by a primer sequence and connected into an expression vector to obtain a recombinant expression vector, the recombinant expression vector is guided into escherichia coli further, and genetically modified recombinant bacteria containing target genes are screened and obtained. According to the glutamine synthetase exogenous expression method based on the bacillus megaterium, the problem that the using effect is limited severely due to the fact that the expression quantity of glutamine synthetase gene sequences in natural bacillus megaterium is very small is solved; and genetically engineered bacteria are used for expressing the glutamine synthetase gene sequences, so that the expression quantity of the gene is increased greatly.

Description

technical field [0001] The present invention relates to a method in the technical field of genetic engineering, specifically a glutamine synthetase gene sequence, a recombinant vector containing the gene, a recombinant vector transformed expression strain, and the use of the recombinant bacteria to produce the expression product of the gene and its application. Background technique [0002] Nitrogen is an essential mineral element with the largest demand for organisms, and it is mainly used for the synthesis of amino acids, nucleotides and other nitrogen-containing compounds necessary for organisms. Nitrogen utilization plays a very important role in agricultural production. my country has now become the world's largest nitrogen fertilizer producer and consumer, and its nitrogen fertilizer usage is still increasing year by year. However, the low utilization rate of nitrogen fertilizer and the environmental pollution caused by excessive use have become a worldwide problem. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/70C12N1/21C12R1/19C12R1/11
Inventor 周培冯海玮孙玉静支月娥刘群录陆伟时唯伟卫星初少华
Owner SHANGHAI JIAO TONG UNIV
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