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Glutamine synthetase gene, protein coded by same and cloning method therefore

A technology of glutamine and gene encoding, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc.

Active Publication Date: 2012-10-31
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the application of inorganic nitrogen fertilizers will cause a series of environmental problems, especially fresh water, and thus the eutrophication of rivers and seawater.

Method used

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  • Glutamine synthetase gene, protein coded by same and cloning method therefore
  • Glutamine synthetase gene, protein coded by same and cloning method therefore
  • Glutamine synthetase gene, protein coded by same and cloning method therefore

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Embodiment 1

[0019] Embodiment one: DvGS2 Cloning and analysis of the full-length coding region of the gene: Our laboratory uses SMART TM The cDNA construction kit constructed 3 Salina cDNA libraries, and obtained the corresponding EST libraries through large-scale sequencing of the cDNA sequences in the libraries (data not published). Through sequence alignment, a salina glutamine synthetase gene was isolated and named as DvGS2 . The corresponding clones of the cDNA library were selected for sequencing, and the cDNA sequence containing the complete ORF of the gene was obtained. The sequencing results showed that: DvGS2 The full length of the cDNA is 1440bp, which contains the specific tailing signal TGTAA and poly(A) structure of the green algae gene. The analysis result of Vector NTI9.1.0 software showed that the gene encoded a protein with 385 amino acids, the molecular weight of the protein was about 42.2kDa, and the isoelectric point was 5.78. Using Euk-mPLoc 2.0 from the Cel...

Embodiment 2

[0020] Embodiment two: DvGS2 Functional analysis in Escherichia coli mutants: by enzyme cleavage method, the enzyme cleavage site is Sal I+ Xba I (these two restriction sites are located on the library plasmid), the DvGS2 The full-length cDNA was excised from the library plasmid to obtain a gene fragment containing the complete ORF.

[0021] Then the cloned target fragment was inserted into E. coli constitutive expression vector pEGFP-1, and transformed into E. 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., E. Flores, and F.J. Florencio, Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene. J Bacteriol, 1992. 174(2): p. 650-4. Due to the deletion of the gene encoding glutamine synthetase, the mutant ca...

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Abstract

The invention relates to a glutamine synthetase gene, a protein coded by same and a cloning method thereof. The gene is from dunaliella, the base sequence of the gene is represented by SEQ ID No.1, and the gene has a function of synthesizing glutamine synthetase and is proved to be capable of obviously improve the glutamine synthesizing capacity of Escherichia coli which is a model organism. Thus, it is indicated that the disclosed full-length gene and the amino acid sequence of the gene have application values in plant gene engineering aspects of improving nitrogen utilization rate of organisms and increasing nitrogen content in organisms.

Description

technical field [0001] The invention relates to a glutamine synthetase gene, its encoded protein and its cloning method. Background technique [0002] Nitrogen is one of the essential nutrients for the growth of organisms, including NO 3 － , NH 4 ＋ And some of their assimilation products are used to synthesize the amino acids and nucleotides required by organisms and a variety of different nitrogen-containing compounds. Nitrogen is also the mineral element that plants demand the most, and the availability of nitrogen sources in the soil is relatively small, which naturally becomes one of the most important factors limiting crop yields. Since exogenous nitrogen is very effective in increasing crop yield, periodic application of inorganic nitrogen fertilizers has become an important way to increase crop yield in concentrated agricultural systems. Many crop varieties are selected by growers in part because of their higher availability of applied nitrogen fertilizers. Howe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52
Inventor 宋任涛范倩岚屈武斐孙晓亮朱晨光许政暟
Owner SHANGHAI UNIV
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