Descurainiasophia cold-resisting gene DsCOR gene, preparation method and application thereof

A technology of Artemisia annua and gene, applied in the field of cold resistance gene DsCOR of Artemisia annua and its preparation, can solve problems such as the research gap of cold resistance gene, and achieve the effects of improving cold resistance, cold resistance and cold resistance.

Inactive Publication Date: 2008-03-19
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The medicinal use, nutrition and health care, and industrial oil production of Artemisi...

Method used

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  • Descurainiasophia cold-resisting gene DsCOR gene, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of Artemisia indica cold resistance gene DsCOR

[0034] 1. Cloning of cold resistance gene DsCOR of Artemisia indica

[0035] (1) The source of Artemisia persicae:

[0036] Seeds of Artemisia sophia (Descurania sophia L) were purchased from Chengdu Seed Company, Sichuan Province;

[0037] (2) Cultivation of materials:

[0038] After the seeds of Artemisia persicae were sterilized, they were sown in the 0 In the culture tank of the culture medium, culture at 25°C for 4 weeks, place the sterile seedlings at 4°C for low temperature stress, wherein the photoperiod is 16h light, 8h dark, and low temperature treatment for 3d;

[0039] (3) PolyA+RNA isolation (PolyA+RNAisolation):

[0040] Use Trizol reagent (Invitrogen) to extract cold-acclimated Artemisia annua total RNA, the extracted total RNA is subjected to 1% non-denaturing agarose gel electrophoresis, and then analyzed its quality by scanning ultraviolet light with a wavelength of 280nm;

[0...

Embodiment 2

[0078] Example 2 Containing the construction of Artemisia indica cold-resistant gene DsCOR recombinant plasmid

[0079] According to the coding sequence of Artemisia persicae cold resistance gene DsCOR of the present invention, design the primer that amplifies ORF, and introduce restriction enzyme site BamHI and SacI respectively on forward primer and reverse primer, also must have on the carrier The same restriction enzyme cutting site, and can make the target fragment inserted into the vector in the correct direction, so as to construct the recombinant plasmid containing the ORF of the cold-resistant gene DsCOR of Artemisia annua. Using the above-mentioned recombinant plasmid as a template, after PCR amplification, the cDNA of the cloned Artemisia persicae cold resistance gene DsCOR was inserted into the plant expression vector pBI121 or p2301G. The restriction sites of the two vectors are the same, both BamH1 and Sac1 , and then the recombinant expression vector was transfo...

Embodiment 3

[0080] Example 3 Preparation of Artemisia indica cold-resistant protein DsCOR

[0081] (1) Construct the prokaryotic expression vector pET32a-DsCOR, and transform Escherichia coli BL21:

[0082] The product amplified by the primers at both ends of the ORF was gel-recovered for the target fragment, that is, the ORF fragment of the COR gene of Artemisia indica, and double-digested with restriction endonucleases BamHI and SacI. The prokaryotic expression vector pET32a was also digested with BamHI and SacI. The carrier and DNA fragments were mixed at a molar ratio of 1:3, ligated with the DNA Ligase Kit Ver.2 kit, and left overnight at 16°C. The ligation product was transformed into BL21 competent cells, and positive clones were selected;

[0083] (2) Induced expression and SDS-PAGE detection of cold-resistant protein DsCOR of Artemisia indica:

[0084] Inoculate the verified Escherichia coli BL21 containing plasmid pET32a-COR into LB medium containing Amp with 1% inoculum amou...

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Abstract

The present invention provides a coding gene of a descurainia sophia cold-resistant protein DsCOR, and a method for producing the cold-resistant protein DsCOR through the recombination technology. The present invention also provides a recombinant plasmid which contains the descurainia sophia cold-resistant gene DsCOR, and a preparation method. The present invention also provides the amino acid sequence of the descurainia sophia cold-resistant protein DsCOR, and the preparation method; on the other hand, the present invention also discloses the application of the descurainia sophia cold-resistant gene DsCOR in the aspects of improving the anti-coldness and cold resistance of the plant.

Description

technical field [0001] The invention relates to an Artemisia indica cold-resistant gene DsCOR and a preparation method thereof, and also relates to the application of a recombinant plasmid containing the Artemisia indica cold-resistant gene DsCOR, which belongs to the technical field of bioengineering. technical background [0002] Temperature is one of the important conditions for the normal growth and survival of plants, and it is also one of the factors that limit the shape and distribution of plants. Plants have a temperature range suitable for their own growth and survival. If the ambient temperature exceeds this range, it will affect the normal growth and development of the plant, causing damage to the plant itself, and even causing the death of the plant. [0003] The damage to plants by low temperature is global, and the economic loss caused by low temperature in the world is very huge every year, which is a very serious natural disaster in the world's agricultural p...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N1/21
Inventor 曹毅乔代蓉白林含徐辉刘玉坤
Owner SICHUAN UNIV
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