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Multi-gene binary expression vector constructed by using homologous recombination and preparation method and application of multi-gene binary expression vector

A binary expression vector, homologous recombination technology, applied in the field of genetic engineering

Inactive Publication Date: 2012-01-25
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method of constructing a multi-gene binary expression vector by gradually adding genes into the T-DNA of the binary expression vector by means of multiple homologous recombination has not yet been reported.

Method used

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  • Multi-gene binary expression vector constructed by using homologous recombination and preparation method and application of multi-gene binary expression vector
  • Multi-gene binary expression vector constructed by using homologous recombination and preparation method and application of multi-gene binary expression vector
  • Multi-gene binary expression vector constructed by using homologous recombination and preparation method and application of multi-gene binary expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Basic carrier acquisition

[0066] Materials used in preferred examples: Pea (Pea ( Pisum sativum L.) Variety pods and big vegetables are materials; E. coli (E. coli ( Escherichia color ) Based DH5α, E. coli XL1-BLUE, root cancer Agrobus ( Agrobacterium tumefaciens ) The strain EHA105 is preserved by this laboratory; PBI121 plasmid and PCAMBIA1302 plasmid is preserved by this laboratory; PCR2.1-Topo carrier (Invitrogen, US), Peasy-T3 carrier (Germany); DNA Marker, carrier PBR322 purchased from Shanghai.Biological Engineering Technology Service Co., Ltd.; PUC18 carrier, PMD18-T carrier, restricted endogenase, Klenow Fragment enzyme, T4-DNA connection enzyme, TAQ DNA polymerase, Primstar HS DNA polymerase purchased from Takara (JAPAN); E. Z. N. A.Plasmid Miniprep Kit and E. Z. N. A. Gel Exteaction Kit are purchased from Omega (USA); ammonia, ampiculin (AMP), Kanamycin (KAN), TETRACYCLINE (TC), Rifampicin, Rifampicin, Rifampicin, Rifampicin.Streptomycin (STR) and bacte...

Embodiment 2

[0092] Basic dual carrier construction

[0093] (1) Construction of PVCT2105 carrier

[0094] Use ECOR I and HIND III enzyme carrier PVCT1215, agarose gel electrophoresis appraisal, abandon 2635 BP fragment, and recycle 2007 BP fragment; cut the PVCT2020 carrier with the same enzyme, agar sugar gel electrophoresis appraisal, discard 51 BP fragments, recycle9481 BP fragment; After purifying the recycling fragment, the T4 DNA is connected to the connection. The reaction condition is 16 ℃ overnight, and the PVCT2105 carrier is obtained.Such as Figure 8 Show, the PVCT2105 carrier contains Pslectin Target gene.

[0095] (2) Construction of the PVCT2120 carrier

[0096] Use PMAC I and HIND III enzyme carrier PVCT2105 to identify the electro -aging gel electrophoretic appraisal, abandon 413 BP and 1118 BP fragments, and recover 9957 BP fragments; use XBA I enzyme to cut PVCT1226, Klenow Fragment enzymes, and then use Hind to use HindIII enzyme cutting, after the appraisal of agar sugar g...

Embodiment 3

[0121] Example 3 The Construction of the Dual Gene Dual expression carrier of homologous reorganization

[0122] Such as Figure 17 It shows that the basic dual -elevated carrier PVCT2121 is used to convert the root cancer EHA 105 to feel the state cells, which contains YEB solid cultivation in RIF (100mg / L), STR (100mg / L) and KAN (50mg / L).Base resistance screening, obtained Agrobacteria EHA105 containing a basic dual -elevated carrier PVCT2121, named EHA105 / PVCT2121.The AMP is based on AMP to screen the intermediate carrier PVCT2125 freezing fusion method to transform EHA105 / PVCT2121 to feel the state cells.The YEB solid medium is selected to obtain EHA105, which is reorganized DNA molecular PVCT2126, which has a reorganized DNA molecule, named EHA105 / PVCT2126, PVCT2126 such as attachment Figure 18 Show.Two homologous recombinant fragments on the PVCT2125 carrier and the PVCT2121 carrier occur in the homologous reorganization in root cancer agricultural bacteria.Since the PVCT21...

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Abstract

The invention relates to the field of gene engineering, in particular to a multi-gene binary expression vector constructed by using homologous recombination. The multi-gene binary expression vector is formed by connecting single genes or multiple genes into a multi-gene polymer through polyclonal enzyme cutting sites so as to form a homologous recombination intermediate vector and adding the single-gene or multi-gene polymer into a basic binary vector by homologous recombination, wherein the homologous recombination can be performed for multiple times, so that the T-DNA length of the basic binary vector is gradually increased. The invention also discloses a construction method of the multi-gene binary expression vector and application in plant transgenes. The multi-gene binary expression vector applied in plant genetic modification can transfer multiple target genes at the same time, and has the advantage of high co-transformation efficiency.

Description

Technical field [0001] The present invention involves the field of genetic engineering, which specializes in the use of homologous reorganization to build a multi -gene dual expression carrier, and also involves the use of homologous reorganization to build a multi -gene dual expression carrier method and its application. Background technique [0002] After the function of many genes is clear, a large number of genes need to be introduced into cultivation crops to improve their comprehensive agronomy or multiple traits.Therefore, large -scale genetic transformation of genes or genetic transformation of large -segment DNA is one of the important development directions for plant transgenic research and utilization.At present, multi -gene conversion plants are mainly through multi -gene aggregation methods, including multiple transformation methods for multiple expression vectors, a multi -gene -allele of the multi -gene monopo carrier. [0003] Multiple transformation methods for m...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/64A01H5/00
Inventor 张兴国苏承刚杜小兵钱春陈友龙谢玉会万发香
Owner SOUTHWEST UNIVERSITY
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