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A plant-mediated RNAi vector for silencing the arginine kinase gene pxak of diamondback moth and its application

A technology mediated by moth arginine kinase, applied in application, plant products, genetic engineering, etc., can solve problems such as application difficulties, and achieve the effect of increasing mortality and reducing pupation rate

Active Publication Date: 2021-08-31
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In early RNAi research, the effect of gene silencing was generally achieved through injection, mixed food feeding, etc., but these methods were difficult to apply in field pest control. In 2007, the first use of transgenic plants to express pest gene dsRNA to Reports on the control of cotton bollworm, that is, plant-mediated insect RNAi control of pests (Mao et al. , 2007)

Method used

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  • A plant-mediated RNAi vector for silencing the arginine kinase gene pxak of diamondback moth and its application
  • A plant-mediated RNAi vector for silencing the arginine kinase gene pxak of diamondback moth and its application
  • A plant-mediated RNAi vector for silencing the arginine kinase gene pxak of diamondback moth and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Plutella xylostella arginine kinase wxya Amplification of full-length gene CDS

[0028] Total RNA was extracted from a single 4th instar larva of Plutella xylostella, the remaining genomic DNA was removed with DNase, and 1 μg of RNA was reverse-transcribed into first-strand cDNA with reverse transcriptase. Using PxAKF / PxAKR as the primer pair (PxAKF: 5'- ATG GTG GACGCT GCA ACT CTT G -3'; PxAKR: 5'- TTA CAG GGA CTT CTC GAT CTT GAT GAG C -3'), high-fidelity polymerase amplified target gene wxya (Genbank accession number: HQ327310.1, 1068 bp). The reaction system is 50 μl: 1 μl DNA polymerase, 1 μl cDNA, 2 μl upstream and downstream primers, 1 μl dNTP, 25 μl buffer buffer, ddH 2 O supplemented to 50 μl. PCR reaction program: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 90 seconds, 35 cycles; extension at 72°C for 5 minutes. The PCR product was detected by agarose gel e...

Embodiment 2

[0029] Embodiment 2: Plutella xylostella arginine kinase gene wxya Plant expression RNAi vector construction of target sequence

[0030] attB 1 -AKF / attB 2 -AKR from plasmid pEASY- wxya amplified gene wxya The corresponding 384bp RNAi target region (SEQ ID NO: 1), and utilize attB 1 -AKF / attB 2 -AKR amplifies the target region, adding attB at both ends of the target region 1 and attB 2 sequence, ie attB 1 -AK-attB 2 (attB 1 -AKF: 5'- ggg gac aag ttt gtacaa aaa agc agg ctc a - GCC AAC GCT TGC CGC TTC TGG -3'; attB 2 -AKR: 5’- ggggac cac ttt gta caa gaa agc tgg gtc – GAT GTC GTA CAC GCC GCC CTC -3’). The PCR reaction system is 50 μl: 1 μl DNA polymerase, 1 μl plasmid template, 2 μl upstream and downstream primers, 1 μl dNTP, 25 μl buffer buffer, ddH 2 O supplemented to 50 μl. PCR reaction program: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 30 seconds, 35 cycles; extension at ...

Embodiment 3

[0031] Example 3: Agrobacterium-mediated carrier pHells-ds AK Transformed Arabidopsis

[0032] The recombinant plasmid pHells-ds constructed by freeze-thaw method AK Transformed into Agrobacterium tumefaciens GV3101 competent cells, spread evenly to LB solid medium containing final concentrations of 25 mg / L rifampicin, 50 mg / L gentamicin and 100 mg / L spectinomycin, Incubate in dark at 28°C for 60 hours to obtain monoclonal colonies, inoculate the monoclonal colonies into 4 mL of LB liquid medium with a sterile toothpick, and culture at 28°C for 15 hours with shaking at 180 rpm to obtain Agrobacterium bacteria liquid. At the same time, Arabidopsis seeds were sown at 23°C, under a photoperiod of 2000 lux of 16 hours of light and 8 hours of darkness, and a humidity of 60%-70%, and Agrobacterium transfection was performed after Arabidopsis grew buds. That is, add 0.1 mL of Agrobacterium bacteria liquid to 100 mL of LB liquid medium, shake and cultivate at 180 rpm at 28°C for 1...

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Abstract

The invention belongs to the field of agricultural biotechnology and relates to a method capable of expressing the arginine kinase gene of diamondback moth in transgenic plants wxya The RNAi vector of the double-stranded RNA (dsRNA) structure is introduced into the plant genome through an Agrobacterium-mediated method, so as to obtain transgenic plants resistant to Plutella xylostella. The present invention shows that transgenic plants can lead to diamondback moth through the inoculation test. wxya The transcriptional level of the gene was down-regulated by 66.5%-79.7%, which reduced the pupation rate and increased the mortality rate by 17.5%, providing a new method for the control of diamondback moth and a new strategy for pest control.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology and relates to a method capable of expressing the arginine kinase gene of diamondback moth in transgenic plants wxya An RNAi vector with a double-stranded RNA (dsRNA) structure is introduced into the plant genome through an Agrobacterium-mediated method to obtain transgenic plants resistant to diamondback moth, providing a new strategy for the control of diamondback moth. Background technique [0002] diamondback moth ( Plutella xylostella L.) is one of the globally important pests on cruciferous vegetables. It is estimated that the economic losses and control costs of diamondback moth to my country are as high as 770 million U.S. dollars per year (Li et al ., 2016). At present, chemical pesticides are still the main means of controlling Plutella xylostella, but the large-scale use of pesticides not only causes harm to other organisms and even humans, but also pollutes the environmen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/65A01H5/00A01H6/20
CPCC12N15/65C12N15/8218C12N15/8286
Inventor 杨广傅淑刘昭霞陈金芝孙庚晓尤民生
Owner FUJIAN AGRI & FORESTRY UNIV
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