Glutamine synthetase gene of acinetobacter as well as encoding protein and cloning method of glutamine synthetase gene

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: 2014-08-06
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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  • Glutamine synthetase gene of acinetobacter as well as encoding protein and cloning method of glutamine synthetase gene
  • Glutamine synthetase gene of acinetobacter as well as encoding protein and cloning method of glutamine synthetase gene
  • Glutamine synthetase gene of acinetobacter as well as encoding protein and cloning method of glutamine synthetase gene

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Experimental program
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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 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 glutamine synthetase gene of acinetobacter as well as encoding protein and a cloning method of the glutamine synthetase gene. The gene is from acinetobacter bacteria and adopts a base sequence represented by SEQ ID NO.1. The gene encodes glutamine synthetase, can play functions of glutamine synthetase in escherichia coli and can recover glutamine synthesis capacity of escherichia coli mutants, and application values of gene in efficient utilization of other biological nitrogen are further predicted simultaneously.

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 Applications(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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