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Gene expression cassette, recombinant expression vector and preparation method and application thereof

A technology of gene expression cassettes and expression vectors, applied in the field of genetic engineering, can solve the problems of plant lethality, unsuitable hairy root screening, etc., and achieve high transformation efficiency

Pending Publication Date: 2021-12-21
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high similarity between the hairy root and the normal adventitious root, how to distinguish the transgenic root from the adventitious root of soybean has become a new problem
In the experiment of transgenic plants, herbicide-resistant genes can be used to screen transgenic plants, but herbicides such as Basta will be transpired in plants through transpiration, which is lethal to some plants above the ground, so it is not suitable for hairy root screening

Method used

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  • Gene expression cassette, recombinant expression vector and preparation method and application thereof
  • Gene expression cassette, recombinant expression vector and preparation method and application thereof
  • Gene expression cassette, recombinant expression vector and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] A method for preparing a recombinant expression vector, comprising the following steps:

[0047] (1) Insert the promoter Gmubi between the CaMVpoly(A) signal and the NOS terminator of the initial vector pCAMBIA1303 to obtain Vector-P;

[0048] (2) insert the egfp reading frame between the promoter of Vector-P obtained in step (1) and the NOS terminator, obtain Vector-P-E;

[0049] (3) Insert the coding sequence of the F2A peptide between the egfp reading frame of the Vector-P-E obtained in step (2) and the NOS terminator, to obtain Vector-P-E-2A;

[0050] (4) between the 2A peptide coding sequence of Vector-P-E-2A obtained in step (3) and the NOS terminator, insert a multi-site artificial linker sequence to obtain the recombinant expression vector Vector-P-E-2A-MCS, the nucleoside of the vector The acid sequence is shown in SEQ NO: 4; the insertion method is double enzyme digestion (connection uses T4 ligase).

Embodiment 2

[0052] A method for preparing a recombinant expression vector, comprising the following steps:

[0053] (1) Insert the promoter GmubiXL between the RB T-DNArepeat and the NOS terminator of the initial vector pBI121 to obtain Vector-P;

[0054] (2) insert the egfp reading frame between the promoter of Vector-P obtained in step (1) and the NOS terminator, obtain Vector-P-E;

[0055] (3) insert the coding sequence of T2A peptide between the egfp reading frame of Vector-P-E obtained in step (2) and the NOS terminator, obtain Vector-P-E-2A;

[0056] (4) Inserting a multi-site artificial linker sequence between the 2A peptide coding sequence and the NOS terminator of Vector-P-E-2A obtained in step (3) to obtain the recombinant expression vector Vector-P-E-2A-MCS; wherein the insertion method is Double digestion (ligation using T4 ligase).

Embodiment 3

[0058] A method for preparing a recombinant expression vector, comprising the following steps:

[0059] (1) Insert the NOS terminator into the MCS of the initial vector pPZP100, and insert the promoter Gmubi between the NOS terminator and LB T-DNArepeat to obtain Vector-P;

[0060] (2) insert the egfp reading frame between the promoter of Vector-P obtained in step (1) and the NOS terminator, obtain Vector-P-E;

[0061] (3) Insert the coding sequence of P2A peptide between the egfp reading frame of Vector-P-E obtained in step (2) and the NOS terminator, obtain Vector-P-E-2A;

[0062] (4) Inserting a multi-site artificial linker sequence between the 2A peptide coding sequence and the NOS terminator of Vector-P-E-2A obtained in step (3) to obtain the recombinant expression vector Vector-P-E-2A-MCS; wherein the insertion method is Double digestion (ligation using T4 ligase).

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Abstract

The invention relates to the technical field of gene engineering, in particular to a gene expression cassette, a recombinant expression vector and a preparation method and application thereof. The invention provides the gene expression cassette. The gene expression cassette comprises a promoter, an egfp reading frame, a coding sequence of 2A peptide and a multi-site artificial linker sequence which are connected in sequence. Compared with the prior art, the vector contains a GFP-2A expression cassette driven by a soybean endogenous promoter, screening of positive transgenic roots by taking GFP as a marker gene is a visual screening mode, the defect that chimera plants are sensitive to herbicides is avoided, transgenic root screening can be carried out, and the positive rate of screened roots can also be ensured.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a gene expression cassette, a recombinant expression vector and a preparation method and application thereof. Background technique [0002] Reverse genetics is one of the important methods for studying gene phenotypes, overexpressing the target gene can identify the phenotype of the gene. The common reverse genetics method is transgenic, and it takes 5 to 8 months to obtain T1 transgenic plants of soybean, and the experiment period is very long. Differences in gene expression in roots can affect root development and resistance. In the study of root biology, researchers often only focus on the phenotype of the root, not the whole plant. Exploring a rapid and specific method for producing transgenic soybean roots can shorten the experimental period. [0003] Studies have pointed out that Agrobacterium rhizogenes K599 can induce soybean to produce hairy roots, and subs...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113C12N15/65A01H5/06A01H6/54
CPCC12N15/8212C12N15/8216C12N15/8217C07K14/415
Inventor 段玉玺杨若巍范海燕陈立杰王惠朱晓峰王媛媛玄元虎刘晓宇
Owner SHENYANG AGRI UNIV
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