Mog-2 Gene of Pine Xylophilus and Its Application in Developmental Disturbance

A technology of mog-2 and pine xylophilus, applied in the fields of application, nematicides, genetic engineering, etc., can solve the problems of embryonic lethality, slow growth of worms, and reproductive system defects

Active Publication Date: 2021-06-08
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mog-2 gene mutants or Caenorhabditis elegans worms disrupted by mog-2 siRNA have defects in the reproductive system, which can lead to slow growth and embryonic lethality

Method used

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  • Mog-2 Gene of Pine Xylophilus and Its Application in Developmental Disturbance
  • Mog-2 Gene of Pine Xylophilus and Its Application in Developmental Disturbance
  • Mog-2 Gene of Pine Xylophilus and Its Application in Developmental Disturbance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Cloning of Gene Sequence of Mog-2 Gene of Pine Xylophilus

[0039] (1) Extraction of Pine Xylophilus DNA

[0040] Take the 25°C pine wood nematode NXY61 strain continuously cultured on the PDA medium covered with Botrytis cinerea, and separate the nematodes by the improved Bellman funnel method, adjust the concentration of the nematode liquid to about 2000 heads / mL, and take about 1mL Put the nematode liquid into a 1.5mL centrifuge tube, centrifuge, rinse, and remove the supernatant for later use;

[0041] Use a homogenizer to homogenize the nematodes in the centrifuge tube, and add 180 μL of Buffer GTL;

[0042] Add 20 μL Proteinase K and vortex to mix the sample thoroughly. 56°C water bath until the tissue is completely lysed;

[0043] Add 200μL Buffer GL, vortex and mix well. Add 200 μL of absolute ethanol, vortex and mix well;

[0044] Add all the solution obtained in the previous step into the absorption column that has been loaded into the collection tube, ce...

Embodiment 2

[0084] Based on the results of Example 1, developmental interference was performed using double-stranded siRNA of the mog-2 gene

[0085] (1) Primer design:

[0086] According to the cDNA sequence of the pine xylophilus mog-2 gene obtained in Example 1, the primer sequence of the double-stranded siRNA used for the interference of the pine xylophilus mog-2 gene was designed and sent to the company for synthesis. The designed and applied double-stranded siRNA is located at the CDS sequence of the mog-2 gene of B. xylophilus, specifically:

[0087] mog-2-siRNA-F 5'-GCUUUGACCAACAACAAUUTT-3'

[0088] mog-2-siRNA-R 5'-AAUUGUUGUUGGUCAAAGCTT-3';

[0089] (2) Interference system

[0090] Filter the plate of pine xylophilus cultured for 5-7 days in an incubator at 25°C for 4-6 hours using the Bellman funnel method, collect the nematode liquid at the bottom of the funnel rubber tube about 4 cm, and pick the 2nd instar larvae of pine wood nematode under a microscope. Rinse with steril...

Embodiment 3

[0096] Based on the results of Example 2, the growth and development of pine xylophilus and the development of offspring were inhibited after the developmental interference of pine xylophilus was interfered with by siRNA.

[0097] After mog-2 gene siRNA interfered with the second-instar larvae of B. xylophilus, the sex ratio of the developed contemporary (F0 generation) adults was 1.21, which was significantly smaller than that of the blank control F0 generation adults (2.6075). There was a significant difference in the ratio of males and females between the control group and the blank control group at the level of P>0.05. (attached image 3 )

[0098] mog-2siRNA can affect the male-female phylogeny of B. xylophilus individuals, make B. xylophilus individuals more masculine, and reduce the sex ratio in B. xylophilus populations.

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Abstract

The invention relates to the technical field of nematode disease prevention and control, in particular to the mog-2 gene of pine xylophilus, and a patent application for using the gene to interfere with the development of pine xylophilus to prevent and control pine xylophilus disease. The full-length DNA sequence of mog‑2 gene of pine wood nematode is 953bp, including 3 introns and 4 exons, and the start codon and stop codon of the DNA sequence are ATG and TGA, respectively. The length of CDS sequence of mog-2 gene of B. xylophilus composed of exons is 843bp. This application has cloned the mog‑2 gene of pine wood nematode and analyzed its function. It is believed that the gene is closely related to the growth and development of pine wood nematode, especially sex determination; using siRNA to interfere with the development of pine wood nematode can cause pine wood nematode Nematodes are obviously masculinized and their population fecundity is reduced. The siRNA can be used to interfere with development to prevent and control pine wood nematode disease.

Description

technical field [0001] The invention relates to the technical field of nematode disease prevention and control, in particular to the mog-2 gene of pine wood nematode, and a patent application for using the gene to interfere with the development of pine wood nematode to prevent and control pine wood nematode disease. Background technique [0002] Background of pine wood nematode disease: Pine wood nematode disease is a worldwide quarantine disease, which has the characteristics of rapid onset, high fatality rate, and difficulty in control. Once introduced, it will cause large-scale death of pine forests. The disease was first discovered in Japan in 1905, and is currently mainly distributed in China, Japan, South Korea, Mexico, Portugal, Canada, the United States and other countries; since it was introduced in 1982, it has gradually spread in the pine forest area of ​​my country. As of 2019, 18 Many districts and counties in this province have been listed as pine wood nematode ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/11C07K14/435A01N57/16A01P5/00
CPCA01N57/16C07K14/4354
Inventor 刘振宇张蒙爱卢园张志凯
Owner SHANDONG AGRICULTURAL UNIVERSITY
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