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Soybean borer lgpgrp-lb gene and its application

A soybean carnivorous insect and anti-eating insect technology can be used in application, genetic engineering, plant genetic improvement, etc., and can solve the problems of polluted environment, low efficiency, and increased drug resistance of soybean carnivorous insects.

Active Publication Date: 2021-09-21
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical insecticides currently used are all broad-spectrum insecticides, which can not only kill target insects, but also kill beneficial insects. At the same time, large-scale and long-term use of chemical insecticides not only increases the resistance of soybean borer insects year by year, but also pollutes the environment
Traditional breeding methods for breeding soybean varieties resistant to soybean borer, although effective, take a long time and are inefficient

Method used

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  • Soybean borer lgpgrp-lb gene and its application
  • Soybean borer lgpgrp-lb gene and its application
  • Soybean borer lgpgrp-lb gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cloning and sequence analysis of the LgPGRP-LB gene of soybean carnivora

[0026] According to the sequencing results of the soybean worm transcriptome (SRR5985986), the transcriptome data was analyzed by blast, and a unigene sequence (c65269.graph_c0) highly homologous to the silkworm PGRP-LB (XM_021349692) was obtained, using ORF finder ( https: / / www.ncbi.nlm.nih.gov / orffinder / ) and DNAMAN software for analysis, and 2 specific primers covering the entire sequence of LgPGRP-LB were designed:

[0027] LgPGRP-LB primer 1:5-ATCATTAAGAGGGGTGGTC-3';

[0028] LgPGRP-LB primer 2:5-CGTACCCTGGGCACTTTT-3'.

[0029] PCR amplification was carried out with the genome of soybean carnivora as a template, pre-denaturation at 95°C for 5 min, 95°C for 1 min, 58°C for 1 min, 72°C for 1 min, 35 cycles, and a final extension at 72°C for 10 min. Carry out PCR reaction with primer 1 and primer 2, and obtain the amplification product from soybean worm cDNA template, and the resu...

Embodiment 2

[0030] Example 2 Analysis of the expression pattern of the LgPGRP-LB gene of soybean carnivore

[0031]In order to determine the expression of LgPGRP-LB gene of soybean carnivore in different developmental stages of soybean carnivore and in different tissues of soybean carnivore larvae, soybeans in four stages of egg, larva, pupa and adult were collected starting from the appearance of soybean carnivore adults in soybean fields at the end of July and early August. For heartworm samples, the collected worm samples were placed in RNase-free EP tubes and immediately frozen in liquid nitrogen, and then stored in a -80°C low-temperature refrigerator. Each experiment was repeated 3 times. The 3-4th instar larvae of Soybean carnivora were selected for dissection, and the dissected 7 tissues of ganglia, epidermis, salivary gland, midgut, ovary, testis and fat body were respectively placed in RNase-free EP tubes and immediately frozen in liquid nitrogen. Store in a low temperature fre...

Embodiment 4

[0037] Example 4 Immune response of LgPGRP-LB to bacterial infection

[0038] In order to determine the recognition of gram-positive and gram-negative bacteria by the LgPGRP-LB gene of soybean carnivore. 90 mature larvae were divided into three groups, and 5 uL of gram-negative bacteria E.coli, gram-positive bacteria B.subtilis and normal saline (CK) were injected intraperitoneally, respectively. Then they were fed with artificial feed, put into an artificial climate box with a temperature of 26 °C, a humidity of 80%, and 18 hours of light, after rearing for 24 hours, the mRNA of the larvae was extracted, and the expression of LgPGRP-LB mRNA was detected by fluorescence quantitative PCR. The results showed that LgPGRP -LB responds to both gram-negative and gram-positive bacterial infections ( Figure 4 ), but the expression level of LgPGRP-LB was significantly increased compared with control larvae after infection with Gram-positive bacteria, which was 2.71 times higher than ...

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Abstract

The invention relates to the field of plant genetic engineering, and provides a soybean borer LgPGRP‑LB gene, the base sequence of which is shown in SEQ ID NO:1. The dsRNA of the soybean borer LgPGRP‑LB gene was synthesized in vitro, and the dsRNA of the soybean borer LgPGRP‑LB gene was introduced into the soybean borer larvae at the end of the first instar by feeding method. Gene silencing thus affected the growth and development of soybean borer larvae, and even caused soybean borer larvae to pupate about ten months earlier. The gene can be used as a target gene to construct an RNAi expression vector for cultivating soybean germplasm with high resistance to the soybean borer.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a soybean carnivore LgPGRP-LB gene and its application. Background technique [0002] Soybean heartworm (Leguminivora glycinivorella (Mats. Obraztsov)) is one of the most serious insect pests in soybean producing areas in Northeast my country, mainly because the larvae bore into the pods and damage the beans. The insect feeding rate is 10% to 20% in normal years, 30% to 40% in severe years, and over 50% in individual years. The damaged soybean kernels are incomplete or broken, and the soybean yield and quality are reduced. Chemical pesticides are a common method of controlling heartworms. The chemical insecticides currently used are broad-spectrum insecticides, which can not only kill target insects, but also beneficial insects. At the same time, the large-scale and long-term use of chemical insecticides not only increases the resistance of soybean carnivores year by ye...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/82C12N5/10A01H5/00A01H6/54
CPCC07K14/43563C12N15/8218C12N15/8286
Inventor 孟凡立宋波袁强曹英雪倪鹤嘉李洋李天宇王占春杨明宇刘新新王国月刘珊珊
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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