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cyp5013c2 protein and its coding gene and Tetrahymena expressing cyp5013c2 protein

A Tetrahymena, gene technology, applied in the application field of degrading DDT, can solve problems such as the increase of DDT concentration

Active Publication Date: 2015-11-25
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, although DDT is mainly used in the southern hemisphere, the concentration of DDT in the northern hemisphere ocean is still rising due to ocean currents and atmospheric effects.

Method used

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  • cyp5013c2 protein and its coding gene and Tetrahymena expressing cyp5013c2 protein
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  • cyp5013c2 protein and its coding gene and Tetrahymena expressing cyp5013c2 protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the growth curve of wild-type Tetrahymena under the treatment condition of different concentration DDT

[0037] 1. Dissolve p,p'-DDT in DMSO to obtain a mother solution with a concentration of 0.1M, and store it in the dark at 4°C.

[0038] 2. Inoculate 2 mL of Tetrahymena thermophila SB210 strain in the early logarithmic growth phase into 20 mL of SPP medium, so that the cell concentration of Tetrahymena thermophila SB210 strain is 3×10 4 cells / mL.

[0039] 3. Adjust the concentration of the mother liquor prepared in step 1 with DMSO and add it to the system obtained in step 2, so that the concentrations of p,p'-DDT are 0.25, 1, 4, 16, 64 or 256 mg / L, and set up inoculation, etc. Volumetric DMSO controls.

[0040] 4. Culture the system obtained in step 3 with shaking at 30°C and 90 rpm, take 200 μL of samples after 0, 3, 6, 9, 12, 24, 36 or 48 hours, count with a cell counter, and make a growth curve.

[0041] Tetrahymena can survive when exposed to DD...

Embodiment 2

[0046] Example 2, Screening genes capable of resisting DDT stress from Tetrahymena

[0047] The CU428 strain of Tetrahymena thermophila was used for genome-wide gene expression microarray analysis, and the gene expression was calculated and analyzed with ArrayStar2.0 software. The same volume of DMSO-treated samples was used as the control group, and the up-regulation or down-regulation of DDT exposure was screened out by 2 times. Among the above genes, it was found that the expression level of CYP5013C2 gene was significantly up-regulated under DDT exposure. The CYP5013C2 protein is shown in sequence 1 of the sequence listing, and the CYP5013C2 gene is shown in sequence 2 of the sequence listing. The amino acid codons in Tetrahymena are different from other species. There are three stop codons (tga, tag and taa) in other species, but there is only one stop codon (tga) in Tetrahymena, and both tag and taa encode glutamine.

Embodiment 3

[0048] Embodiment 3, real-time quantitative PCR detects the relative expression level of Tetrahymena CYP5013C2 gene

[0049] The early logarithmic growth phase (3×10 5 cells / mL) of Tetrahymena thermophila SB210 strain for 24 hours, and the same volume of DMSO was used as the control group.

[0050] The total RNA was extracted and reverse transcribed into cDNA, and the cDNA was used as a template to perform real-time quantitative PCR with a primer pair composed of F1 and R1, and the data was collected with OpticonMonitor3 software. Use the Relative Expression Software Tool (REST) ​​software to analyze the changes in the relative expression level of the target gene after 17SrDNA correction. The mathematical model is based on the difference in the PCR amplification threshold cycle number threshold cycle (Ct) between the sample and the control group. Significance of the expression level (P<0.05).

[0051] F1: 5'-AGTGATTATTGCCTCATTCTTTGG-3';

[0052] R1: 5'-TGTTTCTTCAGTAACCCCTAA...

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Abstract

The invention discloses CYP5013C2 protein and coding gene thereof, and provides tetrahymena expressing the CYP5013C2 protein and an application of the tetrahymena in DDT degradation. According to the present invention, an application of the tetrahymena overexpressing the CYP5013C2 gene in DDT degradation is protected; the CYP5013C2 gene is the gene coding protein (a) or protein (B), wherein the protein (a) is protein comprising an amino acid sequence represented by a sequence 1 in the sequence table, and the protein (b) is the protein formed by carrying out substitution and / or deletion and / or addition of one or a plurality of amino acid residues on the amino acid sequence represented by the sequence 1, has the same function, and is derived from the sequence 1; and the recombinant tetrahymena has the following advantages that: (1) stress response of the CYP5013C2 gene to the DDT is specific and sensitive; (2) the CYP5013C2 genes involves in a DDT biotransformation process in tetrahymena cells; (3) the tetrahymena has an efficient DDT degradation function; (4) the DDT in a water body can be controlled in the bottom end of a food chain; and (5) environment pollution is not generated.

Description

technical field [0001] The invention relates to a CYP5013C2 protein and its coding gene, and provides Tetrahymena expressing the CYP5013C2 protein and its application in degrading DDT. Background technique [0002] DDT (DDT) is an organochlorine insecticide. Since its commercialization in 1942, it has achieved remarkable results in killing mosquitoes and lice, and stopped the epidemic of typhus. Industrial grade DDT contains two types of isomers, 70-80% is p,p'-DDT with active ingredients, and the rest is by-product o,p'-DDT, which will be metabolized into p ,p'-DDE and p,p'-DDD, and o,p'-DDE and o,p'-DDD. Many studies have shown that the sprayed DDT mainly enters the water body through surface runoff, and then adheres to the surface and cilia of lower aquatic organisms such as algae and zooplankton, and is transported through food chains and food webs. DDT finally ends up in higher organisms. Accumulation, causing long-term harm to wild animals and even humans (including ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A62D3/02C12N15/30C12N15/63C12N1/11C07K14/44A62D101/04A62D101/22C12R1/90
Inventor 缪炜冯立芳袁冬霞
Owner INST OF AQUATIC LIFE ACAD SINICA
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