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Method for preparing plutella xylostella RDL subunit knockout strain and application of method

A Plutella xylostella, gene knockout technology, applied in the biological field, can solve problems such as inability to inherit offspring, inability to completely suppress genes, and no research on resistance of Plutella xylostella, so as to monitor drug resistance, improve accuracy, and guide rational use of drugs in the field Effect

Active Publication Date: 2020-07-03
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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  • Method for preparing plutella xylostella RDL subunit knockout strain and application of method
  • Method for preparing plutella xylostella RDL subunit knockout strain and application of method
  • Method for preparing plutella xylostella RDL subunit knockout strain and application of method

Examples

Experimental program
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Effect test

Embodiment 1

[0036] (1) Selection of target sites

[0037] The cDNA and gDNA sequences of Plutella xylostella RDL1 were downloaded from NCBI to determine the distribution of exons. Search for possible target sites through the online website http: / / www.rgenome.net / , and design the target site sequence PxRDL1-sg of the RDL1 gene: 5'-TTAGCGTATAAAAAAAAGGCC-3'(N20; Seq ID NO: 1), located at The fourth exon of the RDL1 gene; the target sequence of the RDL2 gene, PxRDL2-sg: 5'-GCCAGGATGACGGCAGCGCC-3' (N20; Seq ID NO: 2), is located at the first exon of the RDL2 gene. Plutella xylostella RDL subunit gene structure and knockout target see figure 1 .

[0038] (2) Synthesis of sgRNA

[0039] 1) Refer to Guide-it TM sgRNA In Vitro Transcription and Screening Systems User Manual kit (Takara Bio USA, Inc.) Instructions designed sgRNA template amplification primers PxRDL1-gRNAF (SEQ ID NO: 3), PxRDL2-gRNAF (SEQ ID NO: 4). For the structure of operable elements of sgRNA template amplification primer...

Embodiment 2

[0064] Example 2 Plutella xylostella RDL knockout strain construction

[0065] (1) Collect normally eclosion wild-type diamondback moth, put the egg card stained with vegetable juice into the mating box to attract diamondback moth to lay eggs, collect once within 15 minutes, transfer the collected egg card to a clean glass slide for later use .

[0066] (2) The sgRNAs of RDL1 and RDL2 prepared in Example 1 were mixed with Cas9 protein (Cas9-N-NLSNuclease, GenCrispr, Z03388, China), and the mixed system was: 5uL sgRNA (300ng / uL), 3uL Cas9 protein (1ug / uL) uL), 2uL RNase free water, sgRNA and Cas9 protein are mixed in a mass ratio of 1:2. Incubate at 37°C for 15 minutes. 3uL of the incubated mixture was poured into the drawn capillary needle with the tip of a long gun, and injected after opening under the microscope using the parts of the injection instrument. Place the injected egg cards in a box with suitable feed and wait for hatching. The hatch rate was 11% after RDL1 in...

Embodiment 3

[0073] Example 3 Indoor Toxicity Test of Plutella xylostella RDL Subunit Knockout Strain

[0074] Indoor virulence determination of the two mutants and wild-type diamondback moth obtained in Example 2 using fipronil by leaf dipping method (https: / / www.irac-online.org / methods / plutella-xylostella-larvae / ) In the test, the drug concentration was designed between 0.01 mg / L-6 mg / L according to the LC50 of the two drugs against the wild-type diamondback moth. The result is as Figure 6 It was shown that the LC50 of the RDL1 (RDL1KO) knockout strain was 2.29, the RDL2 (RDL2KO) knockout strain LC50 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 was increased by 10 times; after RDL2 knockout, the sensitivity of diamondback moth to fipronil was increased by 4 times. It is concluded that in the normal wild-type Plutella xylostella, the RDL1 subunit performs the main function of GABA recept...

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Abstract

The present invention discloses a method for preparing a plutella xylostella RDL subunit knockout strain and an application of the method. The method for preparing the plutella xylostella RDL subunitknockout strain comprises the following steps: (1) selecting RDL gene knockout targets by using a CRISPR / Cas9 system; (2) conducting in-vitro synthesis of sgRNA; (3) conducting microinjection of the sgRNA and Cas9 proteins into plutella xylostella eggs; and (4) breeding the target-knockout plutella xylostella to otain the plutella xylostella RDL subunit knockout strain. The present invention alsoprovides the application of the plutella xylostella RDL subunit knockout strain in studying resistance of the plutella xylostella. The method breaks through non-inheritance of a RNAi interference technology and constructs the stable hereditary knockout RDL subunit plutella xylostella strain; and fipronil is used to conduct an indoor toxicity test of the prepared plutella xylostella RDL subunit knockout strain, a new method for studying the resistance of the plutella xylostella is provided and the method can also be used to monitor the resistance of plutella xylostella field populations and guide rational drug use in the field.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing an RDL subunit knockout strain of diamondback moth and its application. Background technique [0002] The diamondback moth (plutellaxylostella L.) belongs to the family Lepidoptera and is an important worldwide economic crop pest. Plutella xylostella is more and more harmful to cruciferous vegetables, but it has many generations, serious overlapping of generations, fast reproduction speed, and great difficulty in control. So far, the main means of controlling Plutella xylostella is still the use of chemical agents, but with the extensive use of chemical insecticides, Plutella xylostella has developed serious resistance, making the control more difficult. Therefore, research on pesticide resistance of diamondback moth, research and development of new pesticides, and formulating reasonable control measures have become particularly important. [0003] γ-aminobut...

Claims

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Application Information

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Patent Type & Authority Applications(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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