Arthrobacte glutamine synthetase gene, coding protein and cloning method thereof

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

Inactive Publication Date: 2013-08-07
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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  • Arthrobacte glutamine synthetase gene, coding protein and cloning method thereof
  • Arthrobacte glutamine synthetase gene, coding protein and cloning method thereof
  • Arthrobacte glutamine synthetase gene, coding protein and cloning method thereof

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Embodiment 1

[0023] Embodiment one: Acineb1 Cloning and analysis of the full-length coding region of the gene:

[0024] By referring to Acinetobacter in the NCBI database ( Acinetobacter spp.) the glutamine synthetase gene sequence of the genus Bacteria, designed the primers: Acneb1F: 5'-ATGAGCATGGCGAACAAGGT-3' and Acneb1R: 5'-TTACARGCTRTARTACATAT-3', from soil microbial metagenomic DNA A glutamine synthetase gene was amplified by PCR, named Acineb1 . 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: Acineb1 The full length of the ORF is 1416bp. The analysis result of DNAstar software shows that the gene encodes a protein with 471 amino acids, the molecular weight of the protein is about 53kDa, and the isoelectric point is 5.05. Use the Gneg-PLoc 2.0 of the Cell-PLoc...

Embodiment 2

[0025] Embodiment two: Acineb1 Functional analysis in E. coli mutants

[0026] Through DNA sequencing analysis, select Acineb1 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 sho...

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Abstract

The invention relates to an Arthrobacte glutamine synthetase gene, coding protein and cloning method thereof. The gene of the present invention is derived from Acinetobacter bacteria, and SEQ ID NO1 shows the base sequence. The gene coding glutamine synthetase can perform the functions of glutamine synthetase in the Escherichia Coli, and can recover the capability of Escherichia Coli mutant for synthesizing glutamine, and simultaneously indicates application values in the high efficiency utilization aspect of biological nitrogen.

Description

technical field [0001] The invention relates to an Acinetobacter glutamine synthetase gene, its encoded protein and a 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. Because of this, there is a need today to reduce nitrogen fertilizer use and to find plant genotypes that are more efficient at using nitrogen. Higher nitrogen use efficiency can ensure that the amount of nitrogen fertilizer used can be reduced without reducing yield. [0003] Nitrogen assimilation is a very important physiologic...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N15/10
Inventor 朱晨光宋任涛王佩佩徐腾蛟王伟陆定唐远平梅冰
Owner SHANGHAI UNIV
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