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Gene with asparagine synthetase function, encoding protein and application thereof

An asparagine, protein-encoding technology, applied in the direction of enzymes, introduction of foreign genetic material, enzymes, etc. using vectors

Inactive Publication Date: 2011-07-13
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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  • Gene with asparagine synthetase function, encoding protein and application thereof
  • Gene with asparagine synthetase function, encoding protein and application thereof
  • Gene with asparagine synthetase function, encoding protein and application thereof

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Experimental program
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Embodiment 1

[0030] Embodiment one: DvASN Cloning and analysis of the full-length coding region of the gene: Take advantage of SMART TM The cDNA construction kit constructed three Salina cDNA libraries, and obtained the corresponding EST libraries through large-scale sequencing of the cDNA sequences in the libraries. Through sequence comparison, a salina asparagine synthase gene was isolated and named as DvASN . 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: DvASN The full length of cDNA is 1773bp, and contains poly(A) structure. The analysis results of Vector NTI9.1.0 software showed that the gene encoded a protein containing 591 amino acids, the molecular weight of the protein was about 66.6kDa, and the isoelectric point was 6.11. Using Euk-mPLoc 2.0 from the Cell-PLoc website and ProtComp Version 9.0 from the Softberry website DvASN The...

Embodiment 2

[0031] Embodiment two DvASN Functional analysis in yeast mutants: By enzyme digestion method, the restriction site is EcoRI+XhoI (these two restriction sites are located on the library plasmid), and the DvASN The full-length cDNA was excised from the library plasmid to obtain a gene fragment containing the complete ORF.

[0032] Then insert the cloned target fragment into the yeast overexpression vector pAJ401, and transfer it into the yeast asparagine synthetase mutant DNY41 (Genotype: MATa, asnA, asnB, his4-519, leu2-3, 112, ura3-52) . Due to the deletion of the asnA and asnB genes encoding asparagine synthase, the mutant cannot grow normally in the medium without asparagine. Depend on image 3 As can be seen, DvASN The expression of the yeast mutant DNY41 can grow under the culture condition without adding asparagine. This complementation experiment validates the gene in yeast DvASN The encoded asparagine synthase can perform the function of asparagine synthase ...

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Abstract

The invention relates to a gene with asparagine synthetase function, encoding protein and application thereof. The gene has one of the following nucleotide sequences: 1, a base sequence shown in SEQ ID NO1; and 2, a DNA sequence which possesses over 95 percent of homology with the nucleotide sequence defined in the sequence 1 of a sequence table and encodes the same biological functional protein. The gene has the function of synthesizing asparagine synthetase, can recover the capability of saccharomyces cerevisiae mutant in synthesizing the asparagine synthetase, and simultaneously indicate the application value of the gene in crop nitrogen high-efficiency utilization aspect.

Description

technical field [0001] The invention relates to a gene with the function of asparagine synthase, its encoded protein and its application. Background technique [0002] Nitrogen element is the main component of protein, including NO3-, NH4+ and some of their assimilation products, which are used to synthesize amino acids and nucleotides required by organisms and a variety of nitrogen-containing compounds. It plays an important role in the development of the plant and is the most closely related nutrient element to the yield. Nitrogen is also the mineral element that plants require the most, and the availability of nitrogen sources in the soil is relatively small, which naturally becomes one of the most important limiting factors for crop yields. Because exogenous nitrogen is very effective in improving crop yield, therefore, in the centralized agricultural system, applying inorganic nitrogen fertilizer in stages has become an important way to increase crop yield. Many crop ...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N15/10
Inventor 宋任涛许政暟范倩岚
Owner SHANGHAI UNIV
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