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 increased independent genetic probability and difficulty in obtaining stable transgenic strains, etc., and achieve the goal of constructing The steps are simple, the effect of enhancing resistance and prolonging the green period

Inactive Publication Date: 2008-10-22
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
  • Plant bivalent anti-reverse gene bielement expression carrier
  • Plant bivalent anti-reverse gene bielement expression carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of 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 column centrifugation kit produced by Tsinghua Tianwei Times Company was used for DNA fragment recovery. Escherichia 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 instructions of the kit.

[0037] 2. Experimental content

[0038] The intermediate vector pSAGI was digested with EcoR I and SacI, and the 0.3kb T-Nos fragment was recovered. At the same time, the binary vector p3301 was digested with EcoR I and SacI, and the 11.3kb fragment was recovered as a vector, and the two fragments were ligated with T4-DNA ligase. At the corresponding site, the vector p3301-nos was obtained, and the 5' end of the newly ins...

Embodiment 2

[0072] Example 2 Construction of vector p3301C-nos

[0073]Reagent equipment and experimental procedure are identical with embodiment 1, and experimental content is as follows:

[0074] Digest the cloning vector pUBC with endonuclease Hind III, recover about 3kb fusion gene Pubi-cbf1 fragment; Connected to the Hind III site of p3301-nos to obtain the vector p3301C-nos, in which the fusion gene Pubi-cbf1 is in a clockwise direction, and there is a SacI single enzyme cutting site at the 5' end of the newly inserted T-nos on p3301C-nos point. see Figure 5 , 6.

[0075] The reaction system is Hind III single enzyme system:

[0076] Plasmid 5μl (about 1μg)

[0077] 10×buffer 2 0.5μl

[0078] Hind III 0.5μl (about 5 units)

[0079] wxya 2 O 44μl

[0080]

[0081] Total 50μl

Embodiment 3

[0082] Example 3 Construction of bivalent binary vector p3301IC

[0083] Reagent equipment and experimental procedure are identical with embodiment 1, and experimental content is as follows:

[0084] Cut the intermediate cloning vector pSAGI with endonuclease Sac I, and recover the about 2.7kb fusion gene Psag without T-nos 12 -ipt fragment, while using endonuclease Sac I to digest the carrier p3301C-nos, reclaim the large fragment in the digested product as a carrier, and use the fusion gene Psag without T-nos 12 The -ipt fragment is forward inserted into the SacI site of p3301C-nos to obtain the bivalent binary vector p3301IC. see Figure 7 ,2.

[0085] The reaction system is Sac I single enzyme digestion system:

[0086] Plasmid 5μl (about 1μg)

[0087] 10×buffer 10.5μl

[0088] SacI 0.5μl (about 5 units)

[0089] 10×BSA 0.5μl

[0090] wxya 2 O 43.5 μl

[0091]

[0092] 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 Arabidopsis' cold response gene transcription activation factor gene cbf1 and the Arabidopsis specific senescence gene promote SAG12 regulated iso-pentenyl transferase ipt gene'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 present invention relates to a kind of Agrobacterium binary expression vector used for plant genetic engineering breeding, specifically, it comprises the transcription activator gene cbfl and Arabidopsis thaliana cold response gene regulated by closely linked maize ubiquitin promoter Pubi A bivalent anti-stress gene binary expression vector of the isopentenyl transferase ipt gene regulated by the promoter SAG12 of the specific senescence gene. 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 freeze-resistant plants, such as Arabidopsis, rapeseed, barley, and rye, have a common feature of cold acclimation response, that is, they have the ability to resist freezing after undergoing low temperature exercise from above 0°C to below 10°C . During this process, plants undergo a series of physiological and biochemical changes. Plant ge...

Claims

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Application Information

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Patent Type & Authority Patents(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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