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Plant bivalent anti-reverse gene bielement expression carrier

A binary expression vector and gene technology, applied in the direction of using vectors to introduce foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of difficulty in obtaining stable transgenic strains, increased independent genetic probability, etc., to achieve enhanced resistance Anti-aging, simple construction steps, anti-aging effect

Inactive Publication Date: 2006-04-12
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Co-transformation must be carried out when two functional genes are transferred into plants at the same time. As a result, the independent inheritance probability of the two functional genes in the progeny of the transgenic plant will be greatly increased, and it is difficult to obtain a stable transgenic plant with linked inheritance of the two functional genes. Tie

Method used

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  • Plant bivalent anti-reverse gene bielement expression carrier
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  • Plant bivalent anti-reverse gene bielement expression carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of the vector p3301-Nos

[0035] 1. Experimental materials and kits

[0036] The restriction endonuclease and ligase kits used were all produced by NEB Company, and the DNA fragments were recovered using the column centrifugation kit produced by Tsinghua Tianwei Times Company. E. coli competent cells were purchased from Tsinghua Tianwei Times Company. Plasmid extraction was performed with a kit produced by Tsinghua Tianwei Times Company, and all operating procedures were carried out according to the kit instructions.

[0037] 2. Experimental content

[0038] The intermediate vector pSAGI was digested with EcoRI and SacI, and the 0.3kb T-Nos fragment was recovered. At the same time, the binary vector p3301 was digested with EcoRI and SacI, and the 11.3kb fragment was recovered as a vector. T4-DNA ligase was used to connect the corresponding positions of the two fragments. On the spot, the vector p3301-nos was obtained, and the 5' end of the newl...

Embodiment 2

[0070] Example 2 Construction of vector p3301C-nos

[0071] The reagent equipment and experimental steps are the same as in Example 1, and the experimental contents are as follows:

[0072]The cloning vector pUBC was digested with endonuclease HindIII, and the fusion gene Pubi-cbf1 fragment of about 3 kb was recovered. At the same time, the vector p3301-nos was digested with endonuclease HindIII, and the 11.6kb fragment was recovered as a vector, and the fusion gene Pubi-cbf1 was connected to At the HindIII site of p3301-nos, the vector p3301C-nos was obtained, in which the fusion gene Pubi-cbf1 was in a clockwise direction, and the 5' end of the newly inserted T-nos on p3301C-nos had a SacI single enzyme cleavage site. see Figure 5 , 6.

[0073] The reaction system is HindIII single enzyme system:

[0074] Plasmid 5μl (about 1μg)

[0075] 10×buffer2 0.5μl

[0076] HindIII 0.5μl (about 5 units)

[0077] ddH 2 O 44μl

[0078] Total 50μl

Embodiment 3

[0079] Example 3 Construction of bivalent binary vector p3301IC

[0080] The reagent equipment and experimental steps are the same as in Example 1, and the experimental contents are as follows:

[0081] The intermediate cloning vector pSAGI was digested with endonuclease Sac I, and the fusion gene Psag without T-nos of about 2.7kb was recovered 12 -ipt fragment, digest the vector p3301C-nos with endonuclease SacI at the same time, recover the large fragment in the digested product as a vector, and use the fusion gene Psag without T-nos 12 -The ipt fragment was inserted into the SacI site of p3301C-nos in the forward direction to obtain the bivalent binary vector p3301IC. see Figure 7 ,2.

[0082] The reaction system is Sac I single enzyme digestion system:

[0083] Plasmid 5μl (about 1μg)

[0084] 10×buffer1 0.5μl

[0085] SacI 0.5μl (about 5 units)

[0086] 10×BSA 0.5μl

[0087] ddH 2 O 43.5μl

[0088] Total 50μl

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Abstract

A binary expression carrier of Agrobacterium for culturing the seeds of plant by genetic engineering contains the closely linked corm ubiquitin promoter Pubi regulated Arabidopsisí» cold response gene transcription activation factor gene cbf1 and the Arabidopsis specific senescence gene promote SAG12 regulated iso-pentenyl transferase ipt geneí»s two-valence stress-resistant gene. Its preparing process includes such steps as inserting the T-Nos of sag12-ipt fusion gene into a binary carrier, inserting ubi-cbf1 fusion gene and inserting sag12-ipt fusion gene.

Description

technical field [0001] The invention relates to an Agrobacterium binary expression vector for plant genetic engineering breeding, in particular to the transcriptional activator gene cbf1 of Arabidopsis cold response genes regulated by the tightly linked maize ubiquitin promoter Pubi and Arabidopsis thaliana A bivalent anti-stress gene binary expression vector of the isopentenyl transferase ipt gene regulated by the promoter of the specific aging gene SAG12. The invention also relates to the construction method of the binary vector, which belongs to the field of plant genetic engineering. technical background [0002] Many frost-resistant plants, such as Arabidopsis thaliana, rapeseed, barley, and rye, have a common feature of cold acclimation response, that is, the ability to resist freezing after undergoing low-temperature exercise at temperatures above 0°C and below 10°C. . During this process, plants undergo a series of physiological and biochemical changes. Under low ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/74C12N15/62C12N15/66C12N15/82
Inventor 孙振元韦善君钱永强韩蕾
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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