Sickle alfalfa elongation factor 2MfEF2 as well as coding gene and application thereof

An elongation factor, alfalfa technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of no cloning and so on

Inactive Publication Date: 2013-06-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The EF2 gene has been cloned from a variety of plants, but has

Method used

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  • Sickle alfalfa elongation factor 2MfEF2 as well as coding gene and application thereof
  • Sickle alfalfa elongation factor 2MfEF2 as well as coding gene and application thereof
  • Sickle alfalfa elongation factor 2MfEF2 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Cloning of MfEF2

[0048] 1. Preparation of alfalfa cDNA template

[0049] The alfalfa plants with a seedling age of 8 to 10 weeks were placed in an artificial climate box at a temperature of 5 °C (light intensity was 200 μmol m -2 s -1 ), the light / dark cycle was 12 hours, and the low temperature treatment was performed for 2 consecutive days.

[0050] Extract total RNA: cut mature leaves, add liquid nitrogen to quickly grind into powder, transfer to a 2ml centrifuge tube, add 1.5ml TRE-Trizol (Guangzhou Boli Biotechnology Co., Ltd.) reagent, shake vigorously for 15 seconds, ice bath for 5 minutes . Centrifuge at 10,000 rpm for 5 min, suck the supernatant into a 2 ml centrifuge tube, add 0.3 ml of chloroform, shake vigorously for 15 seconds, and take an ice bath for 10 minutes. Centrifuge at 12,000 rpm for 15 minutes, transfer the upper aqueous phase (≤0.7ml) to a 1.5ml centrifuge tube, add 0.4ml isopropanol and 0.4ml Buffer A (1.2M NaCl, 80mM sodium citr...

Embodiment 2

[0066] Example 2 Construction of MfEF2 plant expression vector pBI-MfEF2

[0067] Double digestion of plasmid pGM-MfEF2 and vector pBI121: The recombinant plasmids pGM-MfEF2 and pBI-121 were double digestion with XbaI and BamHI respectively. For the enzyme digestion system and method, please refer to the instruction manual of TaKaRa company's commercial enzymes. Reaction system (50 μl): 20 μl pGM-MfEF2 (40 ng / l), 2 μl 10× buffer, 2 μl restriction endonuclease XbaⅠ (10 U / μl) and 24 μl sterile deionized water Dig for 2h; add 2 μl of restriction enzyme BamHI (15U / μL), and digest overnight at 30°C. After the reaction, it was incubated at 65°C for 15 minutes to inactivate the restriction endonuclease, and cooled in an ice bath. The binary expression plasmid pBI-121 was double digested by the same method. The double-digested MfEF2 fragment and pBI121 fragment were purified and recovered by DNA gel recovery kit.

[0068] Construction of plant expression vector pBI-MfEF2: Use T4 D...

Embodiment 3

[0074] Example 3 Expression of MfEF2 gene induced by low temperature

[0075] 1. Obtaining alfalfa template under adverse conditions

[0076] Materials and treatment: The alfalfa plants with a seedling age of 8-10 weeks were placed in an artificial climate box at a temperature of 5 °C (light intensity of 200 μmol m -2 s -1 ), both light and dark time were set to 12 hours of light. The treatment was continued for 4 days with low temperature treatment.

[0077] Extraction of total RNA: using the method of extracting total RNA in Example 1, after a certain period of low temperature adversity treatment, take mature leaves, add liquid nitrogen to grind the samples, use TRE-Trizol reagent to extract the total RNA of leaves, and use a spectrophotometer to extract total RNA from leaves. Determine the concentration of total RNA.

[0078] Synthesis of the first strand of cDNA: using the alfalfa RNA prepared above as a template, using PrimeScript TM RT reagent Kit (TaKaRa company) r...

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Abstract

The invention discloses a sickle alfalfa elongation factor 2MfEF2 as well as a coding gene and an application thereof, belonging to the field of plant gene engineering. An MfEF2 gene is obtained, a plant expression carrier is constructed by the obtained MfEF2 gene, a plant tissue is transferred by using the constructed expression carrier, and then a transgene plant is cultivated. The MfEF2 gene is induced at a low temperature; a method for utilizing the MfEF2 gene to cultivate hard plants is provided by the invention; and the freeze resistance and cold resistance of the transgene plant are improved after the gene is overexpressed through the plant.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to an alfalfa elongation factor 2MfEF2 and its encoding gene and application. Background technique [0002] Abiotic stresses in the environment, such as drought, salinity, cold damage and heat damage, affect the growth and development of plants, cause crop yield reduction, and also affect the normal growth and quality of forest trees, fruit trees, flowers, horticultural ornamental plants, and cultivate stress tolerance. The variety of plants is one of the main objectives of the farming industry. However, plant stress tolerance is a quantitative trait involving at least several hundred genes, so it is necessary to isolate tolerance genes from plants with strong stress tolerance. [0003] The elongation factor EF2 encoded by the EF2 gene is a GTP hydrolysis-dependent translocation enzyme, which mediates the translocation of ribosomes during protein synthesis. Each time a GTP is...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/82C12N15/84A01H5/00
Inventor 郭振飞何思健何雪英
Owner SOUTH CHINA AGRI UNIV
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