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Micrococcus glutamine synthetase gene, and encoding protein and clone method thereof

A technology of glutamine and synthetase, which is applied in the field of glutamine synthetase gene, can solve the problems of low utilization rate of nitrogen fertilizer and water environment pollution, etc.

Inactive Publication Date: 2013-07-17
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, the low utilization rate of nitrogen fertilizer and the problem of water pollution caused by excessive application have become a worldwide problem.

Method used

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  • Micrococcus glutamine synthetase gene, and encoding protein and clone method thereof
  • Micrococcus glutamine synthetase gene, and encoding protein and clone method thereof
  • Micrococcus glutamine synthetase gene, and encoding protein and clone method thereof

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Experimental program
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Effect test

Embodiment 1

[0021] Embodiment one: Mico3 Cloning and analysis of the full-length coding region of the gene:

[0022] By referring to Micrococci in the NCBI database ( Micrococcus spp.) the glutamine synthetase gene sequence of bacteria, designed primers: Micoc1F: 5'-ATGTTCACCTCCCCCGCGGA-3' and Micoc1R: 5'-TCAGACCGAGTAGTAGAGCT–3', PCR from soil microbial metagenomic DNA A glutamine synthetase gene was amplified, named Mico3 . 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: Mico3 The full length of the ORF is 1425bp. The analysis result of DNAstar software shows that the gene encodes a protein with 474 amino acids, the molecular weight of the protein is about 52.6kDa, and the isoelectric point is 4.6. Use the Gpos-PLoc 2.0 of the Cell-PLoc website and the ProtComp V...

Embodiment 2

[0023] Embodiment two: Mico3 Functional analysis in E. coli mutants

[0024] Through DNA sequencing analysis, select Mico3 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 tha...

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Abstract

The invention relate to a micrococcus glutamine synthetase gene, and encoding protein and a clone method thereof. The gene provided by the invention is derived from micrococcus bacteria, and has a basic group sequence as shown in SEQ ID NO. 1. The gene encodes glutamine synthetase, and can obviously improve the capacity of mode creature colibacillus in the aspect of synthesizing glutamine, which foreshows that the disclosed full-length gene and the amino acid sequence have application value in the gene engineering aspect of improving the nitrogen utilization efficiency of organisms and increasing nitrogen element content of the organisms.

Description

technical field [0001] The invention relates to a glutamine synthetase gene, its encoded protein and its cloning method. In particular, a micrococcal glutamine synthetase gene, its encoded protein and its cloning method. Background technique [0002] Nitrogen is the mineral element most needed by organisms, and 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, and plant nitrogen metabolism and its regulation have attracted worldwide attention. However, at present, the low utilization rate of nitrogen fertilizer and the water pollution caused by excessive application have become a worldwide problem. Therefore, it is now necessary to reduce the amount of nitrogen fertilizer used, and to find plant strains with higher nitrogen utilization efficiency, so as to reduce the amount of nitrogen fertilizer used without reducing the yield. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N1/21C12N15/10C12R1/265
Inventor 朱晨光王佩佩王伟吴悄沈春磊唐远平梅冰宋任涛
Owner SHANGHAI UNIV
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