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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: 2014-03-19
SOUTHWEST UNIV
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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

[0064] Example 1 Obtaining the basic carrier

[0065] Materials used in the preferred example: Peas ( Pisum sativum L.) Variety Escherichia coli as material; Escherichia coli ( Escherichia coli ) strain DH5α, Escherichia coli XL1-Blue, Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) strain EHA105 was preserved by our laboratory; pBI121 plasmid and pCAMBIA1302 plasmid were preserved by our laboratory; pCR2.1-TOPO vector (Invitrogen, US), pEasy-T3 vector (TransGen, Germany); DNA marker and vector pBR322 were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.; pUC18 vector, pMD18-T vector, restriction endonuclease, Klenow Fragment enzyme, T4-DNA ligase, Taq DNA polymerase, PrimStar HS DNA polymerase were purchased from TaKaRa Company (Japan); E. Z. N. A. Plasmid Miniprep Kit and E. Z. N. A. Gel Exteaction Kit were purchased from Omega Company (USA); Streptomycin (Streptomycin, Str) and bacterial culture medium were purchased from Beijing Ding...

Embodiment 2

[0091] Basic binary vector construction

[0092] (1) Construction of pVCT2105 vector

[0093] The vector pVCT1215 was digested with EcoR I and Hind III, identified by agarose gel electrophoresis, the 2635 bp fragment was discarded, and the 2007 bp fragment was recovered; pVCT2020 vector was digested with the same enzyme, identified by agarose gel electrophoresis, the 51 bp fragment was discarded, and recovered 9481 bp fragment; after purification, the recovered fragment was ligated with T4 DNA ligase, and the reaction condition was 16°C overnight to obtain the pVCT2105 vector. as attached Figure 8 As shown, the resulting pVCT2105 vector contains PsLectin target gene.

[0094] (2) Construction of pVCT2120 vector

[0095] The pVCT2105 obtained by digesting the vector with PmaC I and Hind III was identified by agarose gel electrophoresis, the 413 bp and 1118 bp fragments were discarded, and the 9957 bp fragment was recovered; pVCT1226 was digested with Xba I, filled with ...

Embodiment 3

[0120] Example 3 Construction of Homologous Recombination Multigene Binary Expression Vector

[0121] as attached Figure 17 As shown, the obtained basic binary vector pVCT2121 was transformed into Agrobacterium tumefaciens EHA105 competent cells by freeze-thaw method, and cultured in YEB solid containing Rif (100mg / L), Str (100mg / L) and Kan (50mg / L) Based on the resistance screening, Agrobacterium EHA105 containing the basic binary vector pVCT2121 was obtained, named EHA105 / pVCT2121. Transform EHA105 / pVCT2121 competent cells by freeze-thawing method based on the Amp screening homologous recombination intermediate vector pVCT2125, in the presence of Kan (50mg / L), Amp (100mg / L), Rif (100mg / L) and Str (100mg / L) The Agrobacterium EHA105 containing the recombinant DNA molecule pVCT2126, which undergoes homologous recombination, was screened on YEB solid medium, named EHA105 / pVCT2126, and pVCT2126 is shown in the attached Figure 18 shown. The two homologous recombination fra...

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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 invention relates to the field of genetic engineering, in particular to constructing a multigene binary expression carrier by using homologous recombination, and also relates to a method for constructing a multigene binary expression carrier by using homologous recombination and its application. Background technique [0002] After the functions of many genes are clarified, it is necessary to aggregate and introduce a large number of genes into cultivated crops to improve their comprehensive agronomic traits or multiple traits. Therefore, the large-scale genetic transformation of genes or the genetic transformation of large fragments of DNA is one of the important development directions of plant transgenic research and utilization. At present, the realization of multigene transformation of plants is mainly through multigene polymerization methods, including multiple transformation methods of multiple expression vectors, co-transformation methods of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/64A01H5/00
Inventor 潘宇张兴国苏承刚杜小兵钱春陈有龙谢玉会万发香
Owner SOUTHWEST UNIV
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