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Application of cucumis sativus CsHMGB gene in reducing pesticide residue and relieving pesticide toxicity

A pesticide residue and gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as environmental pollution, endanger human health, and affect quality, and achieve the goal of avoiding environmental pollution, reducing pesticide residues, and improving safety and quality. Effect

Active Publication Date: 2020-08-14
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Propamocarb is a new type, high-efficiency, broad-spectrum carbamate fungicide, which can effectively prevent melon downy mildew, and is a good way to control cucumber downy mildew One of the commonly used pesticides, but due to its volatility and systemic properties, it is easy to accumulate in the cucumber fruit after application to form pesticide residues, and the rest will settle into the soil or diffuse into the atmosphere. On the one hand, it will cause pesticide residues in cucumbers, affecting quality, causing food safety issues; on the other hand, causing environmental pollution and endangering human health
At present, effective ways to reduce pesticide residues in fruits and vegetables are to reduce pesticide input and accelerate the metabolism of pesticides in plants, but exogenous substances increase production costs and even cause secondary pollution

Method used

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  • Application of cucumis sativus CsHMGB gene in reducing pesticide residue and relieving pesticide toxicity
  • Application of cucumis sativus CsHMGB gene in reducing pesticide residue and relieving pesticide toxicity
  • Application of cucumis sativus CsHMGB gene in reducing pesticide residue and relieving pesticide toxicity

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Experimental program
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Effect test

specific Embodiment approach 1

[0025] Embodiment 1: The application of the cucumber CsHMGB gene in reducing pesticide residues and alleviating pesticide toxicity in this embodiment.

[0026] The pesticide described in this embodiment is propamocarb.

[0027] In this embodiment, specific primers (CsHMGB-F: 5'ATGGAAATTATGGAACACAACAG-3', CsHMGB-R: 5'-TCAAGAGATGAAATCTAGAGAGTCA-3') are designed for PCR amplification.

[0028] PCR system: upstream primer (20 μmol L -1 ) 0.5 μL, downstream primer (20 μmol L -1 )0.5μL, 10×PCRBuffer 2μL (including 20mmol·L -1 Mg 2+ ), dNTPs (10mmol·L -1 ) 2 μL, Taq enzyme (2U) 0.2 μL, add ddH 2 0 to 20 μL.

[0029] PCR program: 94°C for 3min; 30 cycles of 94°C for 1min, 54°C for 1min, and 72°C for 1min; 72°C for 10min; store at 4°C.

[0030] Using the online NCBI (http: / / www.ncbi.nlm.nih.gov) database, the sequences homologous to CsHMGB were analyzed and obtained by BLAST alignment. The phylogenetic tree analysis was carried out using MEGA7 software. The phylogenetic tree wa...

Embodiment 1

[0034] Select cucumber low propamocarb residue line 'D0351' (0.014mg / kg) and cucumber high propamocarb residue line 'D9320' (0.171mg / kg) in this embodiment as test materials ('D0351' and 'D9320' Provided by the Cucumber Research Group of Northeast Agricultural University). In the autumn of 2017, the test materials were cultivated at the Horticultural Experiment Station of Northeast Agricultural University, using the conventional ridge cultivation method and unified management. When the melons are ripe at the tenth joint (harvesting stage, 34 days after planting), the plants are sprayed with 400 times of propamocarb hydrochloride solution, and water spray is used as a control, and the degree of spraying to the leaf margin and fruit drops is the degree. And at 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, 96h and 120h, take the peels of the upper, middle and lower parts of the fruit and 2mm thick samples for mixing, quick-freeze in liquid nitrogen, and store at -80°C spare.

[0035] Pr...

Embodiment 2

[0041] Using the cloned full-length primers CsHMGBF and CsHMGBR, and using the CsHMGB open reading frame plasmid as a template, PCR was performed with Taq enzyme to obtain an overhang with A at the 3' end. Further, CsHMGB was ligated with pCXSN-1250 vector by T4 ligase (refer to the manual) to construct the overexpression vector pCXSN-CsHMGB(+).

[0042] The constructed sense and antisense expression vectors and empty vectors were respectively transformed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method. Using the Agrobacterium-mediated genetic transformation technology of cucumber optimized by Wang et al., the cucumber cotyledon nodes of "infected cucumber line L18" were infected, and after co-cultivation, induced differentiation of adventitious buds, elongation of adventitious buds, rooting and domestication of regenerated plants , to obtain resistant individual plants. A concentration of 1 mg / L glufosinate was selected for screening resistant plants to obtain r...

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Abstract

The invention discloses an application of a cucumis sativus CsHMGB gene in reducing pesticide residue and relieving pesticide toxicity, and relates to an application of the CsHMGB gene in the field ofagriculture. From the perspective of genetics, pesticides are metabolized in plants, the safety quality of cucumis sativuss is improved, pesticide residues are reduced, and meanwhile the problems ofenvironmental pollution and the like are avoided. The residual quantity of propamocarb in a cucumis sativus strain for silencing CsHMGB is increased, and the residual quantity of propamocarb in a cucumis sativus strain for overexpressing CsHMGB is reduced. Cucumis sativus is interfered by ROS after being threatened by propamocarb, the overexpressed CsHMGB can improve the activity of antioxidant enzymes SOD, POD, CAT and the like, and the ASA-GSH circulation capacity is improved. The overexpressed CsHMGB induces up-regulated expression of a detoxifying gene and some genes of a glutathione family and a membrane transporter ABC family. The CsHMGB can relieve poison of propamocarb through an antioxidant enzyme system.

Description

technical field [0001] The invention relates to the application of CsHMGB gene in the field of agriculture. Background technique [0002] Cucumber (Cucumis sativus) is one of the main cultivated crops in protected vegetable production in China, with a wide planting area. Due to the special climatic conditions and continuous planting in greenhouses, cucumbers are extremely vulnerable to pests and diseases. Cucumber downy mildew is a devastating disease. [0003] Propamocarb is a new type, high-efficiency, broad-spectrum carbamate fungicide, which can effectively prevent melon downy mildew. It is one of the commonly used pesticides to control cucumber downy mildew, but due to its volatility and Systemic, after application, it is easy to accumulate in the cucumber fruit to form pesticide residues, and the rest will settle to the soil or diffuse into the atmosphere. On the one hand, it will cause pesticide residues in cucumbers, affect the quality, and cause food safety problem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/34
CPCC12N15/8271C07K14/415Y02A50/30
Inventor 辛明秦智伟李胜男周秀艳王蕾
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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