Descurainiasophia DsCBF gene, preparation method and application thereof

A technology of Artemisia indica and genes, applied in the field of Artemisia indica DsCBF cold resistance gene and its preparation, can solve the problems of lack of pertinence, effort, and time, and achieve the effect of drought resistance and salt-alkaline resistance

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

AI Technical Summary

Problems solved by technology

[0004] Traditional plant improvement methods such as hybridization and mutagenesis are time-consuming, laborious, and untargeted. Cloning cold resistance-re

Method used

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  • Descurainiasophia DsCBF gene, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Preparation of Artemisia persicae DsCBF cold resistance gene

[0027] 1. Cloning of the cold resistance gene DsCBF of Artemisia persicae

[0028] (1) The source (Collection) of Artemisia sophia, the seeds of Artemisia sophia were purchased from Chengdu Seed Company, Sichuan Province;

[0029] (2) Isolation of total RNA (RNA isolation)

[0030] Use Trizol reagent to extract the total RNA of Artemisia annua;

[0031] (3) Cloning of Full-length cDNA of DsCBF cold resistance gene of Artemisia persicae

[0032] According to the known conserved amino acid sequences of CBF1, CBF2, and CBF3 of Arabidopsis thaliana; 662 CBFprotein and 663 CBF-like protein of Brassica napus, degenerate primers were designed, and using the principle of homologous gene cloning, the RACE method was used to complete cDNA Long cloning is divided into three stages:

[0033] In the first stage, RT-PCR amplification is performed using degenerate primers and total RNA as a template:

[0034...

Embodiment 2

[0053] Example 2 Construction of Recombinant Plasmid of Artemisia persicae DsCBF Cold Resistance Gene

[0054] According to the full-length sequence of the DsCBF gene of Artemisia indica, primers were designed to amplify its open reading frame, and two restriction enzyme sites BamHI and SacI were respectively introduced into the forward primer and reverse primer to construct the DsCBF gene expression vector; After PCR amplification, the Artemisia persicae DsCBF gene was cloned into the intermediate vector pMD18-T, pMD18-T was digested with restriction enzymes BamHI and SacI, and the DsCBF gene was recovered; the plant expression vector pBI121 was digested with BamHI and SacI, and the pBI121 fragment was recovered ; Insert the DsCBF gene between BamHI and SacI of pBI121 to form the DsCBF gene expression vector pBIcbf; then transfer it into Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 (Pirece, USA), and finally use it to transform the CBF of the model plant defec...

Embodiment 3

[0055] Example 3 Application of the Recombinant Plasmid Expressing the Cold Resistance Gene of Artemisia persicae DsCBF in Arabidopsis

[0056] Use the inflorescence transfection method to transform Arabidopsis thaliana, and transform according to the following steps:

[0057] (1) Planting Arabidopsis until the flowering stage, the soil surface is covered with gauze or cotton cloth;

[0058] (2) remove the terminal inflorescence, to promote the growth of the secondary inflorescence, the plant will grow rapidly in 4-6 days after removing the terminal inflorescence;

[0059] (3) Cultivate Agrobacterium to the logarithmic growth phase at 28°C;

[0060] (4) Centrifuge at 4000rpm, resuspend the bacteria to OD with fresh 5% sucrose solution 600 =0.8; every 2-3 small pots (small pot diameter 9cm) need to be soaked with 400ml-500ml agrobacterium liquid;

[0061] (5) Before dipping, add surfactant Silwet L-77 (500ul / L) and mix well. If Silwet L-77 is too toxic, reduce the concentrat...

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Abstract

The present invention provides a novel DsCBF gene of a descurainia sophia, a nucleotide sequence of the DsCBF gene, and an amino acid sequence coded by the DsCBF gene and a preparation method of the DsCBF gene. The present invention also provides a recombinant plasmid pBIcbf used for expressing the DsCBF gene of the descurainia sophia and the construction method. The present invention also discloses the application of the DsCBF gene in the aspects of improving the cold resistance, anti-drought, anti-salt and alkali of the plant. The present invention is also advantageous to perform the heredity reformation to the economic plant with weak stress resistance and the application to the popularization of the planting.

Description

technical field [0001] The invention relates to a cold-resistant gene of Artemisia indica DsCBF and a preparation method thereof; a recombinant plasmid for expressing the Artemisia indica DsCBF gene and its application in plant cold resistance, drought resistance and salt-alkali resistance. Background technique [0002] Low temperature is one of the main limiting factors of plant yield and geographical distribution on the earth, and the global crop loss due to low temperature reaches hundreds of billions of dollars every year, which has become a common and concerned problem worldwide. [0003] In order to improve the cold resistance of economic plants, expand their cultivated area, and improve the survival ability and yield of economic plants under stress, people have done a lot of research on the mechanism of plant cold resistance. CBF gene, also known as DREB1 transcription factor, itself exists as a master switch for plants to respond to cold stress. When plants are subj...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82
Inventor 曹毅乔代蓉白林含徐辉代旭兰
Owner SICHUAN UNIV
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