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A transgenic Arabidopsis for remediation of PCBS-contaminated soil

A technology of contaminated soil and Arabidopsis thaliana, applied in the field of bioremediation and environmental protection, can solve the problems of plant toxicity, low efficiency of microbial remediation, and insignificant degradation of monohydroxychlorinated biphenyls, so as to improve tolerance and The effect of accumulating ability and improving repairing efficiency

Active Publication Date: 2021-11-19
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing studies have shown that most microorganisms can only degrade low-chlorinated PCBs, and microbial activity is easily affected by environmental conditions, resulting in low microbial remediation efficiency in PCBs-contaminated soil. Therefore, phytoremediation has become a hot spot in the bioremediation of PCBs-contaminated soil.
[0006] (3) Phytoremediation and its limiting factors
[0010] However, further analysis found that the degradation of monohydroxychlorinated biphenyls is not significant, there are a large amount of monohydroxychlorinated biphenyls in plants, which are still toxic to plants, and the threat to the ecosystem still exists

Method used

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  • A transgenic Arabidopsis for remediation of PCBS-contaminated soil
  • A transgenic Arabidopsis for remediation of PCBS-contaminated soil
  • A transgenic Arabidopsis for remediation of PCBS-contaminated soil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: co-expression TfdB+BphC plant screening

[0040] The 2,4-dichlorophenol hydroxylase gene (TfdB) was obtained from a soil metagenomic library. Studies have shown that TfdB enzymes are substrates for chlorophenols, biphenyls, and all indoles except 3-methylindole. Indole substrates all have certain activity. The 2,3-dihydroxyoxygenase (BphC) gene also came from the soil metagenomic library. 2,3-Dihydroxyoxygenase is a key gene that plants lack to open hydroxylated biphenyls.

[0041] (1) Subcloning of TfdB and BphC genes

[0042] The subclones were derived from the 2,4-dichlorophenol hydroxylase TfdB gene (GQ403987.1) and the 2,3-dihydroxybiphenyl dioxygenase BphC gene (GQ403988.1) from the soil metagenome.

[0043] Using the plasmid stored in the laboratory as a template, the primers FBphC and RBphC containing restriction sites were used to amplify the BphC gene; the primers FTfdB and RTfdB were used to amplify the TfdB gene.

[0044] FBphC: 5'-GGAAGATCT...

experiment example 2

[0052] Experimental Example 2 Plant Transformation and Identification of Transgenic Plants

[0053] Use Arabidopsis as the recipient material for genetic transformation, and screen to obtain lines with target traits (co-expression of TfdB+BphC); use PCR and Western blot methods to carry out molecular identification of transgenic plants, and measure the enzyme activity of TfdB or BphC in the plants According to the biological characteristics of the plants, the plants with the strongest p-biphenyl degradation activity among the co-expressed TfdB+BphC plants were selected for subsequent experiments.

[0054] (1) Plant genetic transformation

[0055] Take an appropriate amount of Arabidopsis seeds, place them in the dark at 4°C for 2-3 days, and carry out vernalization; sow them in a mixture of vermiculite and nutrient soil at a volume ratio of 1:1, and cultivate them in a greenhouse at 22°C, with 16 hours of light and 8 hours of darkness; After about 3 weeks of growth, Arabidops...

Embodiment 3

[0066] Embodiment 3: hydroponic simulation restoration test

[0067] Seeds of wild type and transgenic Arabidopsis were sown on 1 / 2 MS solid medium and cultured respectively. After culturing for 15 days, the growth of Arabidopsis in each group was measured. Take Arabidopsis thaliana seedlings that grow well and are similar, and transfer them to 1 / 2MS liquid medium containing 0.05 μg / ml PCB28, and treat 20 Arabidopsis seedlings for each treatment, at 25°C, 14 / 10h alternating light and dark conditions under cultivation. After 7 days, the culture medium and plants were collected, the plant height and fresh weight of the plants were measured, and the difference in the tolerance of wild-type Arabidopsis and transgenic Arabidopsis to PCB28 was analyzed by tolerance index.

[0068]

[0069] Arabidopsis thaliana was washed and placed on tinfoil paper, and dried in an oven at 60°C for 12 hours. The plants were divided into roots and aerial parts, and the amount of dry matter was re...

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Abstract

A transgenic Arabidopsis thaliana for repairing PCBs-contaminated soil, the invention comprises: subcloning TfdB and BphC genes from the soil metagenome; co-expressing TfdB+BphC plants through the construction of plant expression vectors, and plant genetic transformation mediated by Agrobacterium , screening of transgenic positive plants, transgenic plants repairing PCB 28 contaminated soil and other steps; constructing a pathway similar to bacterial degradation of PCBs in Arabidopsis, improving the tolerance of Arabidopsis to PCBs, reducing the limitation of PCB28 on plant growth, and improving The effectiveness of phytoremediation of PCBs-contaminated soil, and the acquisition of new phytoremediation materials for PCBs-contaminated soil; it has a good application prospect in reducing the threat of pollutants to the ecosystem and improving the ecological safety of PCBs phytoremediation.

Description

technical field [0001] The invention belongs to the technical field of bioremediation and environmental protection, in particular utilizing transgenic plants to remediate soil polluted by organic matter. Background technique [0002] (1) The hazards and pollution status of PCBs [0003] Polychlorinated Biphenyls (PCBs) are a class of artificially synthesized environmental heterogeneous substances, which are characterized in that the hydrogen atoms on the biphenyl ring are replaced by 1-10 chlorine atoms, and there are 209 homologues in theory. PCBs have good thermal and chemical stability, oxidation resistance and insulation properties, and have been widely used as insulating media (such as transformer oil) for capacitors around the world. However, because trace amounts of PCBs can cause carcinogenic, teratogenic and mutagenic effects on organisms, and they have long-distance migration, bioaccumulation and refractory properties, PCBs have been approved by the Stockholm Conv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/20B09C1/10
Inventor 王岩任何军张若男张玉芩杨茹杨凤张嘉贺
Owner JILIN UNIV
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