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Recombinant vector as well as construction method and application thereof

A technology of recombinant vector and construction method, which is applied in the field of insect genetic engineering to achieve the effect of simple operation, simple conditions and high specificity

Active Publication Date: 2020-07-24
INST OF FOOD CROPS HUBEI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no method that can effectively study the regulatory mechanism of Ccdc124 gene and its miRNA in N. lugens.

Method used

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  • Recombinant vector as well as construction method and application thereof
  • Recombinant vector as well as construction method and application thereof
  • Recombinant vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Preparation of the 3'UTR region fragment of Ccdc124 gene of brown planthopper:

[0051] Extraction and Detection of Total RNA from Brown Planthopper Tissue

[0052] Trizol reagent (purchased from Invitrogen) was used to extract the total RNA of the brown planthopper (provided by Wuhan University). For the specific extraction steps, refer to the instruction manual of the Trizol reagent. The dissolved RNA was detected by electrophoresis on a 1.5% agarose gel containing ethidium bromide (EB), and the concentration was measured on an RNA concentration analyzer at the same time.

[0053] Design of primers for the 3'UTR region of Ccdc124 gene in brown planthopper

[0054] According to the 3'UTR region of the brown planthopper Ccdc124 gene sequence (ID: XM_022350855.1) in GenBank, PCR primers were designed using PrimerPremier5.0 software, and the Ccdc124 gene amplification primer sequences were as follows:

[0055] Ccdc124-3'UTR-F:5'-ccggctcgagcaaatcacccgagaacccta-3' (SEQ ID...

Embodiment 2

[0074] Example 2 Application of psi-CHECK2-Ccdc124-3'UTR luciferase reporter carrier

[0075] Artificially synthesized candidate miRNAs

[0076] Synthetic miR-8: 5'-ucuguuucagcagugcgagcgg-3' (SEQ ID No.4) and NC negative control: 5'-ugacaaagucgucugcgagcgg-3' (SEQ ID No.5). The above miRNAs and NC controls were synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd.

[0077] Recovery and passaging of Drosophila cells

[0078] Hold the cryopreservation tube containing 1mL of Drosophila cell suspension and quickly put it into a 37°C water bath (the water level should be lower than the cap of the cryopreservation tube) to shake and thaw, transfer it into a 15mL glass core tube containing 4mL of medium prepared in advance and mix well . Centrifuge at 1000rpm for 4min, discard the supernatant, add 1mL medium and blow evenly. Then all cell suspensions were transferred to culture flasks containing 5 mL of dark medium and cultured overnight. Change the medium the next da...

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Abstract

The invention provides a recombinant vector as well as a construction method and an application thereof, and belongs to the technical field of insect genetic engineering. The recombinant vector comprises a dual-luciferase reporter gene vector and a 3'UTR region nucleotide sequence of a nilaparvata lugens Ccdc 124 gene. The construction method of the recombinant vector comprises the following stepsof 1) performing PCR amplification to obtain the 3'UTR region nucleotide sequence of the nilaparvata lugens Ccdc 124 gene by taking the cNDA of nilaparvata lugens as a template; and 2) recombining the 3'UTR region nucleotide sequence of the nilaparvata lugens Ccdc 124 gene into the dual-luciferase reporter gene carrier to obtain the recombinant vector. The recombinant vector provided by the invention can determine the expression activity of the nilaparvata lugens Ccdc 124 gene, is used for screening microRNA for regulating the nilaparvata lugens Ccdc 124 gene, and can also study the regulation mechanism of the predicted microRNA regulated Ccdc 124 gene.

Description

technical field [0001] The invention belongs to the technical field of insect genetic engineering, and in particular relates to a recombinant vector and its construction method and application. Background technique [0002] Brown planthopper (Nilaparvata lugens) ), belonging to the family Delphacidae in the order Homoptera. Mainly distributed in East Asia, Southeast Asia, the South Asian subcontinent, northern Australia and the South Pacific Islands. Rice is the main host of BPH, and some wild rice and barnyardgrass in the field may also become host plants. Generally speaking, under natural conditions, BPH cannot repeat generations on other plants. The brown planthopper likes a hidden and humid environment, and adults and nymphs generally gather in the lower part of the rice bushes. When brown planthoppers occur in large numbers, thousands of insects often gather at the base of each rice pocket. In severe cases, the lower part of the rice plants will turn black, paralyze...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/65C12N15/12C12Q1/6897
CPCC07K14/43563C12N15/65C12N15/66C12N15/85C12N2800/105C12Q1/6897C12Q2525/207
Inventor 闸雯俊游艾青李三和周雷徐华山杨国才刘凯李培德陈志军方国成陈俊孝
Owner INST OF FOOD CROPS HUBEI ACAD OF AGRI SCI
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