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A kind of method for preparing Plutella xylostella rdl subunit knockout strain and its application

A diamondback moth, gene knockout technology, applied in the biological field, can solve the problems of inability to completely suppress genes, inability to inherit offspring, incomplete knockout of position effects, etc., and achieve the effect of improving accuracy

Active Publication Date: 2022-03-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RNAi technology has the advantages of fast, efficient, strong specificity, and easy operation, and plays an important role in the post-genome era, but it has the disadvantages of position effect, temporary and incomplete knockout (Yine et al..2006), and RANi Interference cannot completely inhibit the expression of genes, it has a certain timeliness, and there are transient disadvantages, and it cannot be passed on to offspring (Sun Yuzhang et al., 2016), and it is impossible to conduct genetic research on the population
[0006] At present, there is no report on the preparation of a stable Plutella xylostella RDL subunit knockout strain, and there is no report on the use of RDL subunits to study the resistance of Plutella xylostella

Method used

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  • A kind of method for preparing Plutella xylostella rdl subunit knockout strain and its application
  • A kind of method for preparing Plutella xylostella rdl subunit knockout strain and its application
  • A kind of method for preparing Plutella xylostella rdl subunit knockout strain and its application

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Experimental program
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Effect test

Embodiment 1

[0035] (1) Selection of target sites

[0036] The cDNA and gDNA sequences of diamondback moth RDL1 were downloaded from NCBI, and the exon distribution was determined. Search for possible target sites through the online website http: / / www.rgenome.net / , and design the target sequence of RDL1 gene PxRDL1-sg: 5'-TTAGCGTATAAAAAAAGGCC-3' (N20; Seq ID NO: 1), located in The fourth exon of RDL1 gene; the target sequence of RDL2 gene PxRDL2-sg: 5'-GCCAGGATGACGGCAGCGCC-3' (N20; Seq ID NO: 2), located in the first exon of RDL2 gene. For the gene structure and knockout target of diamondback moth RDL subunit, see figure 1 .

[0037] (2) Synthesis of sgRNA

[0038] 1) Refer to Guide-it TM sgRNA In Vitro Transcription and Screening Systems User Manual kit (Takara Bio USA, Inc.) instructions design sgRNA template amplification primers PxRDL1-gRNAF (SEQ ID NO: 3), PxRDL2-gRNAF (SEQ ID NO: 4). For the structure of the operability element of the sgRNA template amplification primer, see f...

Embodiment 2

[0063] Example 2 Construction of diamondback moth RDL knockout strain

[0064] (1) Collect the normal eclosion wild-type diamondback moth, put the egg card stained with vegetable juice into the mating box to attract the diamondback moth to lay eggs, collect once within 15 minutes, and transfer the collected egg card to a clean glass slide for use .

[0065] (2) The sgRNAs of RDL1 and RDL2 prepared in Example 1 were mixed with Cas9 protein (Cas9-N-NLS Nuclease, GenCrispr, Z03388, China) respectively, and the mixing system was: 5uL sgRNA (300ng / uL), 3uL Cas9 protein (1ug / uL) uL), 2uL RNase free water, sgRNA and Cas9 protein were mixed in a mass ratio of 1:2. Incubate at 37°C for 15min. 3uL of the incubated mixture was poured into the drawn capillary needle with a long pipette tip, and the injection was performed under the microscope with the injection instrument part. The injected egg card is placed in a box containing suitable feed and awaits hatching. The hatching rate was...

Embodiment 3

[0072] Example 3 Indoor virulence test of diamondback moth RDL subunit knockout strain

[0073] Indoor virulence assay of the two mutants obtained in Example 2 and wild-type Plutella xylostella using fipronil by leaf dip method (https: / / www.irac-online.org / methods / plutella-xylostella-larvae / ) In the test, the drug concentration was designed between 0.01mg / L-6mg / L according to the LC50 of the two drugs on wild-type diamondback moth. The result is as Image 6 It was shown that the LC50 of the RDL1 knockout (RDL1KO) line was 2.29, the LC50 of the RDL2 knockout (RDL2KO) line was 0.05, and the wild type (WT) LC50 was 0.22. Compared with the wild type, after RDL1 knockout, the resistance of diamondback moth to fipronil increased by 10 times; after RDL2 knockout, the sensitivity of diamondback moth to fipronil increased by 4 times. This conclusion: in normal wild-type diamondback moth, RDL1 subunit performs the main function of GABA receptor, which is sensitive to fipronil; RDL2 su...

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Abstract

The invention discloses a method for preparing a Plutella xylostella RDL subunit knockout strain and its application. The invention comprises the following steps: (1) a method for preparing a Plutella xylostella RDL subunit knockout strain; (2) synthesizing sgRNA in vitro; (3) microinjecting the sgRNA and Cas9 protein into diamondback moth eggs; (4) cultivating target Point knockout of diamondback moth, to obtain diamondback moth RDL subunit knockout strain. The invention also provides the application of the Plutella xylostella RDL subunit knockout strain in studying the resistance of the diamondback moth. The method of the present invention breaks through the inheritability of the RNAi interference technology, and constructs a stably heritable RDL subunit knockout strain of Plutella xylostella; the indoor toxicity test of the prepared Plutella xylostella RDL subunit knockout strain is carried out through fipronil, It can provide a new method for the resistance research of Plutella xylostella, and can also be used to monitor the resistance of Plutella xylostella field populations and guide the rational use of pesticides in the field.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a RDL subunit knockout strain of Plutella xylostella and its application. Background technique [0002] The diamondback moth (plutellaxylostella L.), belonging to the family Lepidoptera, is an important pest of economic crops worldwide. The damage of diamondback moth to cruciferous vegetables is more and more serious, but its occurrence is many generations, the generations overlap seriously, the reproduction speed is fast, and the control is very difficult. So far, the main means of controlling the diamondback moth is still the use of chemical agents, but with the large-scale use of chemical pesticides, the diamondback moth has developed serious resistance, and the control is more difficult. Therefore, the research on the resistance of diamondback moth, the research and development of new pesticides, and the formulation of reasonable control measures have beco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/89C12N15/90A01K67/033
CPCC12N15/113C12N15/89C12N15/902A01K67/0339C12N2310/20A01K2217/075A01K2227/706A01K2267/03
Inventor 徐汉虹王坤坤林菲李本杰陈冬平
Owner SOUTH CHINA AGRI UNIV
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