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Method for reducing toxicity of Vip3 protein to transgenic plant through gene fusion

A technology of transgenic plants and gene fusion, which is applied in the application field of constructing Vip3 protein fusion genes and constructing insect-resistant transgenic plants, can solve the problems of easy occurrence of albino, slow growth of transgenic plants, and low insect resistance of transgenic plants, and achieves good The effect of anti-insect activity

Active Publication Date: 2013-04-17
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

For example, the researchers found that after a single vip3 gene was transferred into plants, the obtained transgenic plants with high expression of Vip3 protein were slower in growth and prone to albinism
This is because after the Vip3 protein is directly expressed in the transgenic plant, due to the secretion of the signal peptide, the protein aggregates in large quantities and binds to the plant cell membrane to form pores, thereby causing certain damage to the plant cells and affecting their growth and development.
Another consequence of this is that the insect resistance of transgenic plants is not high

Method used

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  • Method for reducing toxicity of Vip3 protein to transgenic plant through gene fusion
  • Method for reducing toxicity of Vip3 protein to transgenic plant through gene fusion
  • Method for reducing toxicity of Vip3 protein to transgenic plant through gene fusion

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the acquisition of fusion gene

[0058] SEQ ID NO: 1 is the amino acid sequence of Vip3 (789aa) Genebank: ABB72459.1; SEQ ID NO: 2 is the amino acid sequence of GFP (241aa) Genebank: AFA52654.1.

[0059] According to the nucleotide sequences corresponding to the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, vip3 and egfp (encoding GFP protein) genes were synthesized by themselves (Shanghai Shengong).

[0060] The egfp-vip3 fusion gene was obtained by gene splicing by overlap extension (SOE) method. details as follows:

[0061] First, the first round of PCR uses the synthetic egfp gene plasmid as a template, using primers egfpPF1: 5’-acg aagctt atgtctagagtgagcaag (plus HindIII) and egfpPR1: 5’- tgttggatctcc cttgtacagctcgtcc amplifies the egfp gene; meanwhile, using the synthetic vip3 gene plasmid as a template, primer Vip3PF1:5'- ggagatccaaca atgaacaagaataatac and Vip3PR1: 5'-acg ggtacc ctacttaatagagac (plus KpnI) amplifies the vip3 gene. Amo...

Embodiment 2

[0075] Embodiment 2, the construction of the rice expression vector of fusion gene

[0076] The entire DNA sequence of the T-DNA vector pCambia1300-p35S-pZmUbi-G10 is shown in SEQ ID NO:5. The vector contains a glyphosate-resistant gene (EPSPS) as the selection gene for transformation, and a set of HindIII and KpnI sites as the cloning site of the target gene. After the fusion gene egfp-vip3 and pCambia1300-p35S-pZmUbi-G10 vectors were digested with HindIII and KpnI respectively, the target fragments were separated by gel electrophoresis, and the new vectors were obtained by ligating the vectors and fragments Called pCambia 1300-p35S-gfp-vip3-pZmUbi-G10. The schematic diagram of vector construction is shown in figure 1 shown.

[0077] Simultaneously, separate vip3 gene and vip3-egfp fusion gene vectors pCambia 1300-p35S-vip3-pZmUbi-G10 and pCambia 1300-p35S-vip3-gfp-pZmUbi-G10 were constructed as controls by the same method as above.

Embodiment 3

[0078] Embodiment 3, rice transformation mediated by Agrobacterium

[0079] The method of obtaining transgenic rice is to use the existing technology (Lu Xiongbin et al., 1998; Liu Fan et al., 2003). The mature and plump "Xiushui 134" seeds were dehulled, and callus was induced as transformation materials. Take the target gene vector pCambia1300-p35S-gfp-vip3-pZmUbi-G10 and pCambia1300-p35S-vip3-gfp-pZmUbi-G10, pCambia1300-p35S-vip3-pZmUbi-G10 and the empty vector pCambia1300-p35S-pZmUbi-G10 The Agrobacterium plate, pick a single colony inoculation ready for transformation with Agrobacterium. Put the callus to be transformed into the appropriate concentration of Agrobacterium solution (containing acetosyringone), let the Agrobacterium bind to the surface of the callus, then transfer the callus to the co-culture medium, and co-culture for 2 to 3 sky. Wash the transformed callus with sterile water, transfer to the selection medium containing 2mM glyphosate, and select and cul...

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Abstract

The invention discloses a method for reducing toxicity of a Vip3 protein to a transgenic plant through gene fusion. The method comprises the following steps: firstly, a C-end of a polypeptide gene is directly connected with an N-end of the gene vip3; and a connecting peptide consisting of four amino acids is added between the C-end and the N-end so as to connect the C-end and the N-end; and secondly, the fusion protein of the fusion gene in plant cell expression is not concentrated on a cell membrane of the plant. The Vip3 protein is any one of Vip3 protein types of a Bt vegetative period insecticide protein, a amino acid sequence in SEQ ID NO:1 and an amino acid sequence which is 70% similar to the SEQ ID NO:1; the polypeptide is a GFP (Green Fluorescent Protein); and the connecting peptide is Gly-Asp-Pro-Thr. The obtained fusion gene is transplanted into plants so as to obtain transgenic zoophobous.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a method for constructing a Vip3 class protein fusion gene and its application in plants to construct insect-resistant transgenic plants. Background technique [0002] Bacillus thuringiensis Bt is the most widely used insecticidal microorganism in the world. It can produce a series of insecticidal toxins during its growth, among which its insecticidal crystal proteins (Insecticidal Crystal Poteins, ICPs) are the most studied and most widely used . However, some important crop pests (such as cutworms and tea geometrids) are not very sensitive to ICPs in practical applications, and due to the irrational use of Bt products, pests such as diamondback moth have produced varying degrees of sensitivity to Bt preparations. Resistance (Zhao et al., 2003; Griffitts et al., 2001). Therefore, finding proteins with different insecticidal action modes from ICPs has important theoreti...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/82A01H5/00
Inventor 沈志成李京
Owner ZHEJIANG UNIV
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