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Calcineurin catalytic subunit gene and application thereof

A technology of calcineurin catalytic subunit and phosphatineurin catalytic subunit, which is applied in the application field of active aluminum, can solve the problems of self-inhibitory domain shedding and reduce the self-inhibition effect of self-inhibitory domain on enzyme activity, and achieve Low technical cost, increased aluminum resistance, and easy operation

Inactive Publication Date: 2014-12-10
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When Ca2+ exists, the combination of B subunit and A subunit will cause the autoinhibitory domain (AID) in the regulatory fragment to fall off from the catalytic active center, thereby reducing the autoinhibitory domain Autoinhibition of enzyme activity

Method used

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  • Calcineurin catalytic subunit gene and application thereof
  • Calcineurin catalytic subunit gene and application thereof
  • Calcineurin catalytic subunit gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1: Cryptococcus terrestris ( C . humicolus ) Cloning and identification of CNA gene of BSLL1-1 strain

[0025] Genomic DNA of Cryptococcus terrestris was extracted (Osper et al., Molecular Biology Experiment Guide (Fifth Edition), Beijing: Science Press, 2008), and sent to Shanghai Human Genome Research Center for genome sequencing to obtain the CNA genome sequence , design primers according to the sequence, the primer sequence is as follows: Forward primer CNA-F: 5'-GGATCCATGACCCTCTCCGGCGACCCAGC-3' (underlined as Bam HI restriction site), reverse primer CNA-R: 5'-AAGCTTTTAGGCAATAGAGTTCTCGG-3' (underlined Han dIII restriction site). Using the cDNA of Cryptococcus terrestris as a template, the designed primers were used to amplify the CNA gene fragment. The PCR reaction conditions were: pre-denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 2 min. After the cycle,...

Embodiment 2

[0026] Example 2: Verification of the interaction between CNA and CaM

[0027] Synthesize CNA polypeptide 611-627: RLAEVISSPTKGGQGER, immunize male rabbits, and prepare CNA antibody;

[0028] 1) Immunization: Select about 3.0kg New Zealand white rabbits. Before immunization, take negative serum from the ear vein, take 1000μg of the polypeptide of the catalytic subunit of calcineurin, dilute it to 1000μl with normal saline, and then add an equal volume of Freund's adjuvant ( Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for the booster immunization). Grind and mix the solution and adjuvant with a mortar to make the solution into a water-in-oil state. The well-mixed immunogen is injected subcutaneously, and generally immunized three times, the first two immunizations are separated by two weeks, and the last immunization is separated by one week. For the first immunization, 2-3 points are injected on the soles of the...

Embodiment 3

[0033] Example 3: Heterologous Expression of CNA Proteins

[0034] Using the cDNA of Cryptococcus terrestris as a template, the CNA fragment was amplified with specific primers CNA-F and CNA-R, and then combined with restriction endonuclease Bam HI / Han The dIII-digested pET-32a vector was ligated and transformed into Escherichia coli BL21(DE3) strain to obtain the recombinant plasmid pET-32a-CNA. extract the plasmid Bam HI / Han dIII double enzyme digestion identification, two bands of the same size as the target band and the vector fragment were detected, such as figure 2 , indicating that the prokaryotic expression vector pET-32a-CNA was successfully constructed. The recombinant plasmid pET-32a-CNA was transformed into Escherichia coli BL21(DE3) to obtain the recombinant strain Escherichia coli BL21(DE3)-pET-32a-CNA containing the recombinant expression plasmid.

[0035] Recombinant Escherichia coli BL21 (DE3)-pET-32a-CNA bacterial strain and control bacterial stra...

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Abstract

The invention discloses a calcineurin catalytic subunit gene which is cloned in a cryptococcus humicolus BSLL1-1 strain. The nucleotide sequence of the calcineurin catalytic subunit gene is shown in SEQ ID NO:1 and is used for coding the protein with an amino acid sequence shown in SEQ ID NO:2; the protein is expressed in escherichia coli, and the calcineurin catalytic subunit gene is further successfully expressed in saccharomyces cerevisiae. According to the calcineurin catalytic subunit gene, the saccharomyces cerevisiae is enhanced in aluminum resistant capacity, genetically engineered bacteria are capable of adsorbing (or absorbing) activated aluminum contained in a culture medium, and the genetically engineered bacteria of the saccharomyces cerevisiae have application prospect of reducing the content of activated aluminum contained in acid soil.

Description

technical field [0001] The present invention belongs to the field of genetic engineering, and specifically relates to an aluminum-resistant gene, a nucleotide sequence and an amino acid sequence, and a heterologous expression of an aluminum-resistant protein encoded by Cryptococcus terrestris encoding a calcineurin catalytic subunit, and relates to a gene containing the gene Recombinant plasmids and engineering strains expressing the aluminum-resistant gene, their preparation and expression methods, and their application of active aluminum in the adsorption environment. Background technique [0002] Acidic soils are widespread in the world, of which the arable area is 179 million hm 2 . The distribution of acidic soil in my country covers 14 provinces and regions, accounting for about 21% of the country's cultivated land (Xiong Yi and Li Qingkui, 1987). Excessive application of nitrogen fertilizer is the main cause of soil acidification in farmland. In addition, the acid ...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/16C12N15/81C12N1/19B01J20/24B09C1/10C12R1/865
Inventor 年洪娟张磊张晶晶陈丽梅
Owner KUNMING UNIV OF SCI & TECH
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