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High-efficiency plant expression vector capable of flexibly replacing regulatory elements and construction method thereof

A technology of plant expression vector and regulatory element, which is applied in the field of high-efficiency plant expression vector and its construction, can solve the problem that the vector lacks plasticity, is difficult to achieve new and special purposes for researchers, and cannot apply regulatory elements or tags to promoters and genes. and other issues to achieve the effect of high compatibility

Inactive Publication Date: 2013-04-24
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The existing entry cloning is to clone the gene or promoter directly into the vector, and it is impossible to apply new regulatory elements or tags to the promoter and gene, and it is impossible to convert the system of labeling proteins into vectors for labeling other molecules (such as RNA) The system, that is, the carrier lacks plasticity, and it is difficult to realize various new and special purposes of researchers

Method used

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  • High-efficiency plant expression vector capable of flexibly replacing regulatory elements and construction method thereof
  • High-efficiency plant expression vector capable of flexibly replacing regulatory elements and construction method thereof
  • High-efficiency plant expression vector capable of flexibly replacing regulatory elements and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Example 1 Construction of Gene Entry Cloning Vector GEC

[0066] Related sequence (SEQ ID No.9) and map according to the present invention ( figure 1 ), synthesized the sequence between the BspHI and ApaI sites by direct DNA synthesis, and then cloned into the pUC19 plasmid to obtain. Then, according to the enzyme cutting sites of the vector map, each tag is directly synthesized by DNA, and then inserted into the corresponding part of the vector by the method of enzyme cutting and ligation, that is, the gene entry clone of various markers is obtained.

[0067] A schematic diagram of the Gene Entry Cloning Vector (GEC) is shown in figure 1 shown. Entry-F and SP6 are two specific sequencing primer sites; attL1 and attL2 are a pair of recombination sites; MCS I and MSC II are multiple cloning sites; ccdB is a bacterial negative selection marker gene; pUC18ori is a bacterial replication initiation site point; CmR is the bacterial chloramphenicol resistance site; each end...

Embodiment 2

[0068] Example 2 Construction of promoter entry cloning vector PEC

[0069] Related sequence (SEQ ID No.10) and map according to the present invention ( figure 2 ), synthesized the sequence between BspH I and Apa I restriction sites by direct DNA synthesis, and then cloned into the pUC19 plasmid to obtain. Then, according to the enzyme cutting site on the vector map, the enhancer is directly synthesized by DNA, and inserted into the corresponding part of the vector by the method of enzyme cutting and ligation, that is, different promoter entry clones are obtained.

[0070] A schematic diagram of the promoter entry cloning vector (PEC) is shown in figure 2shown. Entry-F and SP6 are two specific sequencing primer sites; attL3 and attL4 are a pair of recombination sites; MCS I and MSC II are multiple cloning sites; ccdB is a bacterial negative selection marker gene; pUC18ori is a bacterial replication origin point; CmR is the bacterial chloramphenicol resistance site; each e...

Embodiment 3

[0071] Example 3 Construction of Plant Binary Purpose Vector BDV

[0072] Related sequences (SEQ ID No.11-14) and maps according to the present invention ( Figure 3-Figure 6 ), synthesize the T-DNA sequence (that is, the sequence between LB and RB) by direct DNA synthesis, and then insert it into the corresponding part of the pGreen plasmid by BglII and PacI double enzyme digestion ligation. Then, according to the restriction sites of the vector map, each screening marker gene is synthesized by direct DNA synthesis, and then inserted into the corresponding part of the vector by the method of enzyme digestion and ligation, that is, the plant binary target expression vector of various screening markers is obtained.

[0073] A schematic diagram of the plant expression vector (BDV1) used to express genomic DNA genes is shown in image 3 shown. LB and RB are the left and right boundaries of T-DNA, respectively; attR1 and attR2 are a pair of recombination sites; BarR is a plant g...

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Abstract

The invention relates to a high-efficiency plant expression vector capable of flexibly replacing regulatory elements and a construction method thereof. The high-efficiency plant expression vector is composed of three vectors: gene entry cloning vector GEC, promoter entry cloning vector PEC and plant double-element target vector BDV, wherein the GEC and PEC respectively carry one set of site-specific recombinant sequence SSRs, the SSRs carried by the GEC are attL1 and attL2, the SSRs carried by the PEC are attL3 and attL4; the BDV carries two set of SSR sites, one set of SSRs are attLR1 and attLR2 which can respectively carry out site specific recombination with the attL1 and attL2, and the other set of SSRs are attLR3 and attLR4 which can respectively carry out site specific recombination with the attL3 and attL4; and the high-efficiency plant expression vector is obtained by carrying out LR reaction on the GEC, PEC and BDV in a reaction system. The high-efficiency plant expression vector provided by the invention has universality, and can be quickly and efficiently combined with any promotor, gene and various regulatory elements to implement various research purposes.

Description

technical field [0001] The invention relates to the construction of a plant expression vector, in particular to a high-efficiency plant expression vector capable of flexibly replacing regulatory elements and a construction method thereof. Background technique [0002] Although significant progress has been made in plant genomics research, and genome sequences of many species of plants have been sequenced, the functions of genes are still poorly understood. The functional evolution and differentiation of phenotypes is similarly poorly understood. Similarly, although the functions of certain plant genes and the interaction between genes have been deeply understood, their detailed functions and interactions under specific conditions are poorly understood. [0003] Gene expression is the first step in the expression of gene function, and the level of gene expression often determines the strength of the corresponding phenotype of the plant. Limiting the expression of the target...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
Inventor 傅永福王旭朱金龙张晓玫
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI