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Method for raising insect resistance of plants

A technology of plant and ability, applied in the fields of plant bioengineering and plant improvement genetic engineering, which can solve the problems of the decline of insect resistance of transgenic insect-resistant plants and the proliferation of other pests

Active Publication Date: 2012-10-17
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Various genetically engineered insect-resistant plants have been found to be less resistant to insects over time, and by exclusively suppressing one pest in the field, other pests begin to infest

Method used

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  • Method for raising insect resistance of plants
  • Method for raising insect resistance of plants
  • Method for raising insect resistance of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0167] Embodiment 1, be used to construct the sequence fragment of dsRNA carrier

[0168] With Asian cotton bollworm (Helicovepa armigera) cDNA library (ZAP express) (contain 1mgg -1 The total RNA of the midgut of the fifth instar cotton bollworm after one day of artificial diet with the concentration of gossypol was used as a material to construct a cDNA library) as a template, and 29 EST fragments for constructing dsRNA expression vectors were amplified, called WXM-1~WXM-29 . The genes related to EST fragments are shown in Table 2.

[0169] Table 2

[0170]

[0171] In the present invention, coding sequences of trypsin, dopa decarboxylase, NADH dehydrogenase flavoprotein 2, ATP synthase β subunit, myosin heavy chain subtype B, and RTM intron sequences are used. Among them, the darkened part in gray is the fragment used to construct the dsRNA vector.

[0172] SEQ ID NO: 1 (WXM-2, trypsin precursor coding sequence, GenBank: EE399600.1):

[0173] ACCCTACTATTGCGGCTCTTT...

Embodiment 2

[0201] Embodiment 2, separation of gene fragments such as trypsin, dopa decarboxylase

[0202] With Asian cotton bollworm (Helicovepa armigera) cDNA library (ZAP express) (contain 1mgg -1 The total RNA of the midgut of the fifth instar cotton bollworm after one day of artificial diet with the concentration of gossypol was used as a material to construct a cDNA library), and PCR amplification was performed to obtain EST fragments for constructing dsRNA expression vectors respectively. The reaction solution is: 5 μL 10× buffer, 2.5 μL 10mMdNTP, 2 μL primer F, 2 μL primer R, 2 μL cDNA, 1 μL pfu enzyme, add water to make up to 50 μL. The PCR reaction conditions were 94°C for 4min, 30 cycles at 94°C for 30s, 55°C for 30s, 72°C for 2min, and 72°C for 10min. DNA fragments of expected size were recovered after PCR product electrophoresis.

[0203] Using cotton bollworm wxm-12 (NADH dehydrogenase ubiquinone flavoprotein 2) as a template, the cDNA sequence of the corresponding gene of...

Embodiment 3

[0236] Embodiment 3, vector construction and Agrobacterium transformation

[0237] 1. Vector Construction

[0238] The structure of the dsRNA vector to be constructed is as follows: figure 1 Shown in A, including the plant expression promoter CaMV35S promoter, a forward (i.e. Sense, S) gene fragment, an internal element of the Arabidopsis RTM gene (i.e. Intron, about 128bp, and a reverse gene fragment (namely Antisense, AS) and NOS terminator. It is obtained by inserting the sequence containing Sense-Intron-Antisense into the BamHI and SacI sites on the pCambia2301 vector (purchased from Cambia Company).

[0239] Using the Arabidopsis genome as a template, first use the internal element (about 128bp) of the Arabidopsis RTM gene (AT2G43730) with the specific primer RTM+(5'-TCTAGAACGTTGTAAGTCTATTTTTG-3'(SEQ ID NO: 27)) containing XbaI and NotI and RTM-(5'-GCGGCCGCTCTGCTGGGTCCAAATCACA-3'(SEQ IDNO:28)) were amplified by high-fidelity enzyme pfu, the PCR product was double-digest...

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Abstract

The invention relates to a method for raising the insect resistance of plants. The invention provides target genes from insects, a construct is designed based on nucleic acid sequences of the target genes and interfering RNA molecules which interfere target gene expression are formed in the plants, after the plants are eaten by insects, the expression of related genes can be inhibited and is less or not affected by the digestive system in insects and other barriers. The method of the invention can raise the insect resistance of plants, reduce the usage of pesticides, reduce the agricultural production cost, and preserve the ecological environment.

Description

technical field [0001] The invention belongs to the fields of plant bioengineering and plant improvement genetic engineering. Specifically, the present invention relates to a batch of new target genes in insects. The expression of these target genes in insects can be specifically inhibited by plant-mediated RNAi technology, thereby inhibiting the growth of insects and improving the insect resistance of plants. The invention discloses the sequences of these target genes and their application in the field of RNAi-mediated transgenic insect resistance. Background technique [0002] In agricultural production, insect pests have always been an important factor affecting crop yields. People have to invest a lot of manpower and material resources every year to suppress pests and increase crop yields. Extensive use of pesticides has caused severe environmental pollution and severely damaged farmland biodiversity. Pesticide residues have gradually become an important factor threate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/84A01H5/00C12N15/11C12N5/10
CPCC12N15/8218C12N15/8286Y02A40/146C12N15/113
Inventor 陈晓亚武秀明毛颖波杨长青王凌健
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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