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Gene DvCRP1 with Cd<2+> and Cu<2+> resistance as well as coding protein and application thereof

A gene coding and gene technology is applied to the gene DvCRP1 with anti-Cd2+ and anti-Cu2+ functions, its encoded protein and its application fields, which can solve the problems of lack of transformation system and inability to study salina

Inactive Publication Date: 2012-12-19
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on salina lacks a transformation system and cannot be studied by mature genetic methods

Method used

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  • Gene DvCRP1 with Cd&lt;2+&gt; and Cu&lt;2+&gt; resistance as well as coding protein and application thereof
  • Gene DvCRP1 with Cd&lt;2+&gt; and Cu&lt;2+&gt; resistance as well as coding protein and application thereof
  • Gene DvCRP1 with Cd&lt;2+&gt; and Cu&lt;2+&gt; resistance as well as coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Cloning and analysis of the full-length coding region of DvCRP1

[0041] The yeast expression library was transformed into the yeast mutant Δyap1 by LiAc heat shock method, in the presence of 150 μM Cd 2+The yeast transformants with mutant phenotype recovery were screened under the screening pressure to obtain candidate clones. In order to exclude false positives caused by spontaneous mutations in the yeast genome, the expression vectors carried by the candidate clones were isolated from yeast cells, transformed into Escherichia coli for amplification, and the amplified vectors were reverted to the yeast mutant Δyap1 and incubated in 150 μM Cd 2+ The clones that can still grow stably under the treatment are the true positive clones. The insert fragment in the expression vector carried by the positive clone was sequenced to obtain the partial cDNA sequence of DvCRP1.

[0042] In order to obtain the cDNA sequence of the full-length coding region, the present ...

Embodiment 2

[0043] Example 2 Sequence analysis of DvCRP1

[0044] The full-length cDNA sequence of the DvCRP1 gene was searched for homology in the corresponding database of NCBI using Blastx, tBlastx and Blastn programs successively ( http: / / blast.ncbi.nlm.nih.gov ), did not obtain effective Blast search results, indicating that the Salina DvCRP1 gene and its homologous genes have not been cloned and studied at present, and are discovered for the first time with anti-Cd 2+ Functional new genes.

Embodiment 3

[0046] Example 3 Functional Analysis of DvCRP1 in Yeast

[0047] (1) Construction of yeast expression vector

[0048] The full-length ORF fragment of DvCRP1 was amplified by using the cDNA clone of DvCRP1 as a template by PCR method. In order to construct the clone, with the help of primer introduction method, an EcoRI restriction site is added to the 5' end of the target sequence, and an XhoI restriction site is added to its 3' end. The primer sequences are as follows:

[0049] DvCRP1+: 5'-attgaattcatgaacggagacgagtgtagg-3'

[0050] DvCRP1-:5'-attctcgagtcagcatctgcacagatcac-3'

[0051] The product amplified by PCR was directional cloned into the vector pAJ401 using the enzyme sites EcoRI and XhoI, and transformed into Escherichia coli Top10. The positive clone pAJ401-DvCRP1 was identified by enzyme digestion and sequencing.

[0052] (2) Transform yeast

[0053] Using the LiAc heat shock transformation method: the empty vector pAJ401 and the expression vector pAJ401-DvCRP1 ...

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Abstract

The invention relates to a gene DvCRP1 with Cd<2+> and Cu<2+> resistance as well as a coding protein and application thereof. In the invention, a yeast functional complementary system is adopted for screening to obtain the gene DvCRP1, an RACE technology is utilized to obtain a full-length cDNA sequence, and the gene is proved to be capable of obviously improving the cadmium and copper resistanceof a model organism yeast and obviously enhancing the accumulation amount of the cadmium in yeast cells. The full-length gene and an amino acid sequence, disclosed in the invention, are found to be the unique cadmium and copper resistant genes of Dunaliella for the first time, and have application values to improving the cadmium and copper resistance of an organism, enhancing accumulation amount of cadmium and copper in the organism and other genetic engineering aspects.

Description

technical field [0001] The invention relates to gene DvCRP1 with anti-cadmium and anti-copper functions, its encoded protein and application. Background technique [0002] With the development of modern industry, heavy metal pollution is becoming more and more serious. Environmental and health problems caused by heavy metal pollution have been reported in many countries. Cadmium (Cd 2+ ) is one of the most toxic heavy metals, and Cd 2+ In the environment, it has strong chemical activity, high mobility and long-lasting toxicity. Since the 1920s, due to the rapid development of mining, smelting, electroplating, chemical and other industries and the discharge of wastewater, water and soil Cd 2+ Pollution is getting worse. Contaminated Cd in water and soil 2+ It is easily absorbed and accumulated by aquatic organisms and higher plants, affecting their growth and metabolism, resulting in a decline in the yield and quality of agricultural products and damage to the ecologica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/10C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C07K14/405
Inventor 宋任涛许政暟孟祥宗
Owner SHANGHAI UNIV
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