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A method for improving the tolerance and degradation ability of plants to polycyclic aromatic hydrocarbons
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A technology of polycyclic aromatic hydrocarbons and tolerance, applied in the field of environmental science, can solve the problems of few, etc., and achieve the effect of safe and efficient expression, improving tolerance and degradation ability
Inactive Publication Date: 2015-08-19
SHANGHAI ACAD OF AGRI SCI +1
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High-molecular-weight PAHs are relatively stable in the environment, and there are few reports on degrading bacteria that can metabolize them as the only carbon source and energy source
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Embodiment 1
[0110] Example 1, Effects of Transgenic Arabidopsis on Phenanthrene and Pyrene Tolerance and Degradability
[0111] 1) Preparation of Agrobacterium Competent Cells
[0112] The Agrobacterium strain is Agrobacterium tumefaciens EHA105, or LBA4404, or GV3101 strain (Biovector Co., LTD). Pick a single bacterium and culture it overnight in 25ml YEB medium (added with 50mg / l rifampicin), transfer 5ml of the bacterial liquid to 100ml YEB medium (added with 50mg / l rifampicin), and cultivate to OD600=0.7-0.8, Place the bacterial solution on ice for 10 minutes, centrifuge at 5000rpm for 10min, 4°C, collect the bacterial cells, add 100ml sterile double distilled water to wash twice. Add 4ml of 10% glycerol to suspend the bacteria and transfer to a 50ml centrifuge tube. Centrifuge at 5500rpm for 10min at 4°C. Collect the bacteria, add 500μl 10% glycerol to suspend the bacteria, and transfer to a 1.5ml centrifuge tube.
[0113] 2) The bivalent gene plant expression vector pCYPUGT was ...
Embodiment 2
[0125] Embodiment 2, the impact of transgenic rice on the degradation ability of phenanthrene and pyrene
[0126] 1) Agrobacterium-mediated transformation of rice
[0127] The rice callus was transformed with the Agrobacterium strain containing the expression vector pCYPUGT obtained in step 2) of Example 1. N6 medium is the basic medium, shelled rice seeds, and immature embryos 12-15 days after pollination are surface-sterilized and inoculated into N6D2 medium to induce callus (N6 medium, hydrolyzed milk protein 500mg / L, Sucrose 30g / L, 2,4-D2mg / L, Phytogel 2.5g / L, pH 5.8); after 4-7 days of culture, callus was taken for transformation. After the Agrobacterium containing the expression vector pCYPUGT was cultured at OD0.8-1.0, centrifuge at 5000g for 8 minutes, wash once with double distilled water, suspend and infect with an equal volume of MS culture medium for 8 minutes, and blot dry the culture medium placed in MSO+NAA1+BA2 in 22°C for 3 days. Then transfer to selection ...
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Abstract
The invention discloses a method for improving tolerance and degradation capacity of a plant to polycyclic aromatic hydrocarbon. The method comprises the particular steps that a reconstructed P450 monooxygenase gene and a synthetized poplar UDP-glucoside glucuronyl transferase (UGT) gene construct a bivalent gene plant expression vector, and then the bivalent gene plant expression vector is mediated and inverted into the plant by agrobacterium. With the adoption of the method, the tolerance and the degradation capacity of the obtained transgenic plant to polycyclic aromatic hydrocarbon are improved, and planting of the transgenic plant facilitates restoring a soil environment polluted by polycyclic aromatic hydrocarbon.
Description
technical field [0001] The invention belongs to the field of environmental science, and in particular relates to a method for improving the tolerance and degradation ability of plants to polycyclic aromatic hydrocarbons. Background technique [0002] Polycyclic aromatic hydrocarbons (PAHs) are mostly petroleum, coal and other fossil fuels, and hydrocarbon-containing substances such as wood, natural gas, gasoline, heavy oil, organic polymer compounds, paper, crop straw and tobacco, which have been incompletely burned or in a reducing atmosphere. produced by thermal decomposition. High-molecular-weight PAHs are relatively stable in the environment, and there are few reports on degrading bacteria that can metabolize them as the only carbon source and energy source. The mineralization of polycyclic aromatic hydrocarbons with four rings and above by most microorganisms generally starts in the way of co-metabolism. In the process of co-metabolism, microorganisms preferentially u...
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