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Application of a gene derived from Klebsiella pneumoniae

A pneumonia bacteria and gene technology, applied in the field of environmental pollutant treatment, can solve problems such as limited efficiency of plants in processing environmental pollutants

Active Publication Date: 2021-04-23
CHONGQING ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention intends to provide an application of a gene derived from Klebsiella pneumoniae to solve the technical problem of limited efficiency of plants in treating environmental pollutants

Method used

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  • Application of a gene derived from Klebsiella pneumoniae
  • Application of a gene derived from Klebsiella pneumoniae
  • Application of a gene derived from Klebsiella pneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Gene optimization and cloning

[0036] Klebsiella pneumoniae was genetically screened to obtain a gene with a sequence such as SEQ ID NO: 1. The protein encoded by the gene has the activity of degrading environmental pollutant TNT, and it was named TNT degrading gene. The gene can be used to construct transgenic plants, and the transgenic plants containing the gene can be used in the practice of phytoremediation of the environment polluted by TNT. The gene derived from Klebsiella pneumoniae of sequence such as SEQ ID NO:1 is transformed according to the plant codon preference, and the optimized gene (called the optimized TNT degrading gene) of sequence such as SEQ ID NO:3 is obtained, and the gene SEQ ID NO:3 Both ID NO: 1 and optimized gene SEQ ID NO: 3 encode a protein with a sequence such as SEQ ID NO: 2. The optimized gene was synthesized by chemical synthesis, and the primers used in the synthesis were as follows:

[0037] First primer: 5'-GGATCCATGGAC...

Embodiment 2

[0051] Embodiment 2: Preparation of transgenic Arabidopsis

[0052] Agrobacterium tumefaciens LBA4404 strain was selected, and SBSV was transferred into the strain by electric shock method. In addition to the LBA4404 strain, one of LBA4404, GV3101 or AGL-1 can be used. The method for preparing Agrobacterium competent cells is a conventional method in the prior art, specifically: first expand and cultivate Agrobacterium, pick LBA4404 bacterial strain single bacterium colony to 25mL YEB medium (all contain 50mg / L rifamycin in the medium ) to cultivate overnight, and then take 5mL of the overnight culture and transfer it to 100ml YEB medium for expanded culture (OD 600 The culture was terminated after reaching 0.8). The bacterial solution was placed on ice for 10 minutes to stop the growth of Agrobacterium, and then the bacterial cells were collected by centrifugation (4°C, 5000 rpm), and the collected bacterial cells were washed twice with sterile double distilled water. Then...

Embodiment 3

[0055] Embodiment 3: the preparation of transgenic tobacco

[0056] Tobacco (K326) is transgenicly manipulated by using the recombinant plasmid SBSV, and the method is the Agrobacterium co-transfection method in the prior art. Tobacco seeds were first sterilized (using 75% alcohol and sodium hypochlorite solution in turn to sterilize), then spread the seeds on MS medium (28°C cultivation temperature), cut off the young leaves of tobacco after germination, and put them in a medium containing 1 μg / mL NAA and 4 μg / mLBA MS medium, cultured at 22°C for 1 day. Get the transgenic Agrobacterium prepared in Example 1 and expand the culture to an OD value of 1.0, centrifuge and collect the thalline, and wash the thalline with sterile water, suspend the thalline with MS culture solution (thalline equal volume), and use the suspension of the thalline The young tobacco leaves were soaked in the liquid for 8 min, then the young tobacco leaves were taken out, the liquid was blotted dry with...

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Abstract

The present invention relates to the field of environmental pollutant treatment, in particular to the application of a gene derived from Klebsiella pneumoniae, the gene is used in the treatment of pollutant TNT, the sequence of the gene is as SEQ ID NO: 1 shown. Optimizing the codons of the gene of the present invention, then constructing an expression vector, and overexpressing the gene in plants can improve the plant's ability to degrade and remove TNT, and improve the plant's resistance and tolerance to TNT stress. This scheme can be used to solve the technical problem of limited efficiency of plants in treating environmental pollutants, and this gene can be applied to the practical operation of environmental restoration and TNT treatment.

Description

technical field [0001] The invention relates to the field of environmental pollutant treatment, in particular to the application of a gene derived from Klebsiella pneumoniae. Background technique [0002] The compound TNT (2,4,6-trinitrotoluene) produced by large-scale production and processing of ammunition has caused serious environmental pollution. Both TNT and its transformation products are toxic. TNT has been identified as a potential carcinogen (Category C) by the U.S. Environmental Protection Agency. Direct contact can cause anemia, liver dysfunction, skin irritation, or damage to the immune system. Therefore, it is very important to remediate TNT-contaminated soil and water. In the exploration and practice of remediation of TNT-contaminated environments, biological methods use microorganisms and plants with potential remediation capabilities to clean the environment, which has gradually become a new remediation method in recent years. Phytoremediation is one of th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/26C12N15/82A01H5/00A01H5/10A01H6/20A01H6/82A01H6/46
CPCC07K14/26C12N15/8259C12N15/8271
Inventor 游双红谭平武峥史文景方波伊洪伟陈元平杨丽张生
Owner CHONGQING ACAD OF AGRI SCI
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