Construction method of plant dual-gene co-expression vector

A technology of co-expression vector and construction method, which is applied in the field of construction of plant dual-gene co-expression vector, can solve the problems of affecting the efficiency of metabolic pathways, and the expression does not tend to be balanced, and achieves the effect of simplifying the screening procedure.

Inactive Publication Date: 2013-05-08
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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Problems solved by technology

However, there is a translation polarity effect in this system, and the expression of each gene does not tend to be balanced. This phenomenon is especially obvious when there are many genes in series, which will seriously affect the efficiency of the entire metabolic pathway.

Method used

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  • Construction method of plant dual-gene co-expression vector
  • Construction method of plant dual-gene co-expression vector
  • Construction method of plant dual-gene co-expression vector

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Embodiment Construction

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0028] When implementing the present invention, take the construction of the co-expression vector of HbWRKY11 and HbWRKY7 as an example for further description, and the specific steps are as follows:

[0029] 1. Design primers

[0030] 1) Design primers, SP-F and SP-R, according to the spacer sequence to be amplified, and introduce Ncol and EcoR Ⅰ restriction sites into the upstream primer and downstream primer, respectively;

[0031] 2) According to the structural characteristics of the multiple cloning site of the pXCS-HAStrep vector, design linkers SP-A1 and SP-A2.

[0032] 2. Vector construction

[0033] 1) Using the vector pGWB605 (GenBank accession number AB543114) as a template and SP-F / SP-R as primers, the spacer sequence was obtained by PCR amplification, and the product size was 341bp;

[0034] 2) Ligate the amplified prod...

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Abstract

The invention relates to a construction method of a plant dual-gene co-expression vector. The construction method is characterized by comprising the following steps of: adding restriction enzyme cutting sites of Nco1 and EcoR I incision enzymes at two ends of a spacer sequence in a plasmid pGWB 605 (the registration number of which is AB543114 in GenBank) through PCR (Polymerase Chain Reaction) amplification, and then cloning the spacer sequence into a pXCS-HAStrep plasmid to construct the plant dual-gene co-expression vector pXCS-Dgene-HAStrep. The co-expression vector only has 5682Kb and can be loaded with two larger foreign genes which are independently expressed in an eukaryotic expression system. A bar gene is used as a screening marker gene in the co-expression vector. According to the construction method, the screening operation of a transgenic plant is simple, and the efficiency is high.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for constructing a plant double-gene co-expression vector. Background technique [0002] In the past, plant transgenics mostly used a single target gene to improve individual traits of plants, but the transformation of a single target gene could not meet the needs of plant improvement, especially the genetic modification of some metabolic pathways or quantitative traits. With the deepening of plant genetic engineering and molecular biology research, multi-gene transformation research came into being and developed rapidly. [0003] At present, the multi-gene co-expression system for plant genetic transformation usually includes a multi-plasmid co-transformation system and a single vector expression cassette system for multiple genes. In the multi-plasmid co-transformation system, multiple foreign genes are respectively cloned into different resistan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/64
Inventor 安泽伟翟琪麟李雅超赵建文谢黎黎高新生李维国黄华孙
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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