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Bacillus subtilis glutamine synthetase gene as well as encoding protein and cloning method thereof

A technology of Bacillus subtilis and glutamine, applied in the direction of biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of water environment pollution and low utilization rate of nitrogen fertilizer

Inactive Publication Date: 2013-07-10
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Nitrogen utilization plays a very important role in crop production. Plant nitrogen metabolism and its regulation have attracted worldwide attention. The problem of low nitrogen utilization rate and water pollution caused by excessive nitrogen fertilizer application is also a worldwide problem.

Method used

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  • Bacillus subtilis glutamine synthetase gene as well as encoding protein and cloning method thereof
  • Bacillus subtilis glutamine synthetase gene as well as encoding protein and cloning method thereof
  • Bacillus subtilis glutamine synthetase gene as well as encoding protein and cloning method thereof

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Embodiment 1

[0021] Embodiment one: GrassBs Cloning and analysis of the full-length coding region of the gene:

[0022] By referring to Bacillus subtilis in the NCBI database ( Bacillus subtilis ) glutamine synthetase gene sequence, designed the primers, the primers are: BsF: 5'-ATGGCAAAGTACACTAGAGA-3' and BsR: 5'-TTAATAYTGAGACATATACT-3', PCR amplified from soil microbial metagenomic DNA A glutamine synthetase gene, named GrassBs . The gene was inserted into the cloning vector pMD18-T, the corresponding positive clone was selected, and the full-length DNA sequence of the gene was carried out to obtain the DNA sequence containing the complete ORF of the gene. The sequencing results showed that: GrassBs The full length of the ORF is 1335bp. The analysis result of DNAstar software shows that the gene encodes a protein with 444 amino acids, the molecular weight of the protein is about 50.3kDa, and the isoelectric point is 4.99. Use the Gpos-PLoc 2.0 of the Cell-PLoc website and the Pr...

Embodiment 2

[0023] Embodiment two: GrassBs Functional analysis in E. coli mutants

[0024] Through DNA sequencing analysis, select GrassBs Gene ORF transcription direction and lactose promoter on vector pMD18-T lacZ The clones with the same transcription direction were extracted and transformed into Escherichia coli mutant ET6017 (Genotype: F-,[araD139]B / r,Δ(argF-lac)169,flhD5301,Δ(fruK-yeiR) 725 (fruA25), relA1, rpsL150 (strR), Δ (glnG-glnA) 229, ha-10, deoC1). See literature: Merida, A., Flores E., Florencio F.J., Regulation of Anabaena sp. strain PCC7120 glutamine synthetase activity in a Synechocystis sp. strain PCC6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene. J Bacteri 1992, 174(2): 650-654. Due to the deletion of the gene encoding glutamine synthetase, the mutant cannot grow normally in the medium without glutamine. This strain was purchased from E. coli Genetic Stock Center, Yale University. Depend on Figure 4 and Figure 5 It shows...

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Abstract

The invention relates to a bacillus subtilis glutamine synthetase gene as well as an encoding protein and cloning method thereof. The bacillus subtilis glutamine synthetase gene is from bacillus subtilis, and the sequence of the bacillus subtilis glutamine synthetase gene is a base sequence shown in SEQ ID NO. 1. Glutamine synthetase encoded by the gene can be used for remarkably improving the glutamine synthesis capability of model organism escherichia coli. The disclosed full-length gene and the amino acid sequence have application values in improvement of the utilization rate of nitrogen of organisms and increase of the content of nitrogen in the organisms in genetic engineering.

Description

technical field [0001] The invention relates to a glutamine synthetase gene, its encoded protein and its cloning method, in particular to a subtilis glutamine synthetase gene, its encoded protein and its cloning method. Background technique [0002] Nitrogen is the mineral element that organisms must and needs the most. It is used to synthesize amino acids, nucleotides and various nitrogen-containing compounds required by organisms. Nitrogen utilization plays a very important role in crop production. Plant nitrogen metabolism and its regulation have attracted worldwide attention. The low utilization rate of nitrogen fertilizer and water pollution caused by excessive application of nitrogen fertilizer are also worldwide problems. [0003] Glutamine synthetase is a key enzyme in the nitrogen assimilation process of plants. The role of GS in plant nitrogen nutrient metabolism is studied by plant genetic engineering methods, and the expression level of GS in nitrogen assimil...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/10C12N15/63
Inventor 朱晨光沈春磊王伟叶先敏王珊珊唐远平梅冰宋任涛
Owner SHANGHAI UNIV
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