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Acinetobacter glutamine synthetase gene, its encoded protein and cloning method

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

Inactive Publication Date: 2016-04-27
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Acinetobacter glutamine synthetase gene, its encoded protein and cloning method
  • Acinetobacter glutamine synthetase gene, its encoded protein and cloning method
  • Acinetobacter glutamine synthetase gene, its encoded protein and cloning method

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Effect test

Embodiment 1

[0021] Embodiment one: Acineb1 Cloning and analysis of the full-length coding region of the gene:

[0022] Acinetobacter ( Acinetobacter spp.) are mainly distributed in water bodies and soils, and are easy to survive in humid environments. Collect a small amount of river bank soil samples, take 1 gram of soil, and extract its metagenomic DNA. By analyzing the glutamine synthetase gene sequence of Acinetobacter bacteria in the NCBI database, the degenerate primers were designed: Acneb1F: 5'-ATGAGCATGGCGAACAAGGT-3' and Acneb1R: 5'-TTACARGCTRTARTACATAT-3'. Using this pair of primers, a glutamine synthetase gene was amplified by PCR from the extracted soil microbial metagenomic DNA, named as 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 ...

Embodiment 2

[0023] Embodiment two: Acineb1 Functional analysis in E. coli mutants

[0024] 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 Anabaenasp.strainPCC7120glutaminesynthetaseactivityinaSynechocystissp.strainPCC6803derivativestrainbearingtheAnabaenaglnAgeneandamutatedhostglnAgene.JBacteriol,1992,174-2:450. Due to the deletion of the gene encoding glutamine synthetase, the mutant cannot grow normally in the medium without glutamine. The strain was purchased from E. coli Genetic Stock Center, Yale University. Depend on Figure 4 and Figure 5 It shows that Acineb1 can complement the g...

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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 physiological ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N15/10
Inventor 朱晨光王欣珍陆定王伟唐远平梅冰宋任涛
Owner SHANGHAI UNIV
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