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Plant insect-resistant gene mVip3Aa, vector and application thereof

A pest-resistant gene and plant technology, applied in plant genetic improvement, botanical equipment and methods, applications, etc., can solve the problems of increased ratio of high-quality transformants, less agronomic traits of transgenic plants, etc., and achieve good resistance

Active Publication Date: 2020-03-24
LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the transgenic plants highly expressing the mVip3Aa protein have fewer agronomic traits, the ratio of high-quality transformants increases, and the pollen is less affected, which effectively solves the negative impact on plants after transferring the Vip3Aa gene

Method used

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  • Plant insect-resistant gene mVip3Aa, vector and application thereof
  • Plant insect-resistant gene mVip3Aa, vector and application thereof
  • Plant insect-resistant gene mVip3Aa, vector and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The acquisition and synthesis of embodiment 1mVip3Aa gene

[0027] 1. Obtain the nucleotide sequence of mVip3Aa

[0028] The amino acid sequence (818 amino acids) of mVip3Aa insecticidal protein, as shown in SEQ ID NO:1 in the sequence listing; The mVip3Aa nucleotide sequence (2457 nucleotides), as shown in SEQ ID NO:3 in the sequence listing.

[0029] The amino acid sequence (1155 amino acids) of Vip3Aa insecticidal protein, as shown in SEQ ID NO: 2 in the sequence listing; Vip3Aa nucleotide sequence (3468) corresponding to the amino acid sequence (1155 amino acids) of said Vip3Aa insecticidal protein coding nucleotides), as shown in SEQ ID NO:4 in the sequence listing.

[0030] 2. Synthesize the above mVip3Aa nucleotide sequence

[0031] The mVip3Aa nucleotide sequence (as shown in SEQ ID NO: 3 in the sequence listing) and the Vip3Aa nucleotide sequence (as shown in SEQ ID NO: 4 in the sequence listing) were synthesized by Nanjing KingScript Biotechnology Co., Ltd....

Embodiment 2

[0032] Embodiment 2 vector construction

[0033] 1. Construction of cloning vector

[0034] The synthesized mVip3Aa nucleotide sequence was connected to 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 LP01-T , its construction process is as follows figure 1 Shown (in which 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 start codon; mVip3Aa is mVip3Aa nucleotide sequence (SEQ ID NO: 3)).

[0035]Then, the recombinant cloning vector LPO1-T was transformed into Escherichia coli T1 competent cells (Transgen, Beijing, China; Cat. No: CD501) by heat shock method, and the heat shock conditions were: 50 μ...

Embodiment 3

[0043] Embodiment 3 Recombinant expression vector transforms Agrobacterium and detects

[0044] (1) Transformation of Agrobacterium with recombinant expression vector

[0045] The recombinant expression vectors LP-PT-03 and LP-PT-03CK that have been constructed correctly were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat.No: 18313-015) by liquid nitrogen method, and the transformation conditions were as follows: : 100 μL Agrobacterium LBA4404, 3 μL plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; inoculate the transformed Agrobacterium LBA4404 in LB test tubes at a temperature of 28 ° C and a rotation speed of 200 rpm Cultivate under conditions for 2 hours, spread on LB plates containing 50 mg / L Rifampicin and 50 mg / L Kanamycin until positive single clones grow, pick single clones for culture and extract their Plasmid, the recombinant expression vectors LP-PT03 and LP-PT03CK we...

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Abstract

The invention discloses a synthetic insecticidal gene mVip3Aa for transgenic insect-resistant plants and a protein for insect-resistant plants. The amino acid sequence of the protein is SEQ ID NO: 1,and the nucleic acid sequence of the coding gene is SEQ ID NO 3. The invention also discloses corresponding vectors and applications of proteins and genes in insect resistance. After the modified mVip3Aa gene of the invention is transferred into a plant, the negative effects of high expression of Vip3Aa gene on plant traits are significantly reduced in the obtained transgenic plant with high expression of mVip3Aa protein, the ratio of high-quality transformants is increased, and the pollen is less affected. Meanwhile, the obtained transgenic plants with high expression of mVip3Aa protein havethe resistance to Mythimna separata, which is better than that with Vip3Aa.

Description

technical field [0001] This application relates to the technical field of genetic engineering biological control, in particular to the artificially modified insect-resistant gene mVip3Aa, its expression vector and its application. Background technique [0002] Biological control is the use of certain beneficial organisms or biological metabolites to control the population of pests to achieve the purpose of reducing or eliminating pests, such as Trichogramma or Beauveria bassiana to control meadow borers. It is characterized by being safe for humans and animals, less polluting to the environment, and can achieve long-term control of some pests; however, the effect is often unstable, and the same investment is required regardless of the severity of meadow moth occurrence. [0003] In order to solve the limitations in the practical application of agricultural control, chemical control, physical control and biological control, scientists have discovered through research that by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/32C12N15/32C12N15/82A01H5/00A01H6/46
CPCC07K14/32C12N15/8286C12N2830/36
Inventor 贾志伟李树秀李晓娇吕玉平李涛赵丽媛张原王强刘枫
Owner LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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