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Method for inhibiting or killing dichocrocis punctiferalis and application thereof

A peach borer and reaction technology, which is applied in the field of inhibiting or killing peach borer, can solve the problems of food security threat, yield reduction, weakening of crop growth potential, etc., and achieves the effect of excellent anti-peach borer characteristics.

Pending Publication Date: 2022-01-11
LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the biotic stress (such as diseases and insect pests, etc.) and abiotic stress (such as drought, cold damage, and salt damage) faced by agricultural production have weakened the growth of crops and reduced yields, posing a huge threat to global food security.
However, there is no relevant report on the control of the peach borer pest by expressing the Cry1Da_7 gene

Method used

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  • Method for inhibiting or killing dichocrocis punctiferalis and application thereof
  • Method for inhibiting or killing dichocrocis punctiferalis and application thereof
  • Method for inhibiting or killing dichocrocis punctiferalis and application thereof

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Experimental program
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Effect test

Embodiment 1

[0025] The acquisition and synthesis of embodiment 1Cry1Da_7 gene

[0026] The Cry1Da_7 gene (SEQ ID NO:1) was synthesized by Nanjing GenScript Biotechnology Co., Ltd., encoding the amino acid sequence (1166 amino acids) of the Cry1Da_7 insecticidal protein shown in SEQ ID NO:2. The 5' end of the synthesized Cry1Da_7 nucleotide sequence is connected with an NcoI restriction site, and the 3' end is connected with an EcoRI restriction site.

Embodiment 2

[0027] Embodiment 2 vector construction

[0028] 1. Construction of cloning vector

[0029] The synthesized Cry1Da_7 nucleotide sequences were respectively connected into the cloning vector pEASY-T5 (Transgen, Beijing, China, CAT: CT501-01), and the operation steps were carried out according to the instructions of the pEASY-T5 vector produced by Transgen Company to obtain the recombinant cloning vector LP20- T, whose construction process is as follows figure 1 Shown (where Kan+ represents the kanamycin resistance gene; Amp+ represents the ampicillin resistance gene; pUC origin represents the replication region sequence of the plasmid pUC, which can guide the double-stranded DNA replication process; LacZ is the LacZ initiation codon; Cry1Da_7 is Cry1Da_7 nucleotide sequence (SEQ ID NO: 1).

[0030] The recombinant cloning vector LP20-T was transformed into Escherichia coli T1 competent cells (Transgen, Beijing, China; Cat. No: CD501) by heat shock method. The transformation ...

Embodiment 3

[0035] Embodiment 3 Recombinant expression vector transforms Agrobacterium and detects

[0036] (1) Transformation of Agrobacterium with recombinant expression vector

[0037] The recombinant expression vector LP-PT20 that has been constructed correctly was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat.No: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 μL Agrobacterium LBA4404 and 3 μL plasmid The DNA (recombinant expression vector) was frozen in liquid nitrogen for 10 minutes, and then water bathed at 37°C for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated in a centrifuge tube containing LB liquid medium and cultured on a shaker at 28°C and 200 rpm 2h, spread on LB solid medium containing 50mg / L rifampicin (Rifampicin) and 50mg / L kanamycin (Kanamycin) until positive single clone grows, pick single clone culture and extract its plasmid, use Restriction endonucleases NotI and SalI digested the recombin...

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Abstract

The invention relates to a method for inhibiting or killing dichocrocis punctiferalis and application thereof, in particular to a method for inhibiting or killing dichocrocis punctiferalis and a method for detecting whether a Cry1Da-7 gene is inserted into a plant genome or not. The method for inhibiting or killing dichocrocis punctiferalis comprises the following steps that (1) a plant of which a genome is inserted into a gene for coding Cry1Da-7 protein as shown in SEQ ID NO: 2 is provided, wherein the plant expresses the Cry1Da-7 protein and generates dichocrocis punctiferalis resistance; and (2) the plant is in contact with dichocrocis punctiferalis, and the dichocrocis punctiferalis is inhibited or killed. It is found that after the Cry1Da-7 gene is inserted into plant genome DNA, the plant shows good dichocrocis punctiferalis resistance, and the insecticidal rate of the Cry1Da-7 gene to dichocrocis punctiferalis reaches 92%-100%.

Description

technical field [0001] The invention relates to the field of insect-resistant genes, in particular to a method and application for inhibiting or killing the peach moth borer. Background technique [0002] At present, the biotic stress (such as diseases and insect pests, etc.) and abiotic stress (such as drought, cold damage, and salt damage) faced by agricultural production have weakened the growth of crops and reduced yields, posing a huge threat to global food security. Among them, insect pests are one of the main biological stress factors affecting agricultural and forestry productivity. For example, the peach borer (Dichocrocis punctiferalis (Guenee)), also known as the peach borer, belongs to the family Lepidoptera and is an omnivorous pest. It is widely distributed in my country and harms crops such as corn, sorghum, peaches, persimmons, and chestnuts Waiting for fruit trees. The grain loss caused by peach borer is huge every year. [0003] As the environmental probl...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/32A01H5/00A01H5/10A01H6/46C12Q1/6895C12N15/11
CPCC12N15/8205C12N15/8286C07K14/325C12Q1/6895C12Q2600/13
Inventor 李晓娇马小伟吕玉平贾志伟王建海石小堰李涛李树秀王杰林黎彦
Owner LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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