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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: 2015-05-20
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 not been cloned from the very cold and drought tolerant Medicago sativa

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] Cloning of embodiment 1MfEF2

[0048] 1. Preparation of Medicago cDNA template

[0049] The 8-10-week-old Medicago sativa plants were placed in an artificial climate chamber at a temperature of 5°C (light intensity of 200 μmol m -2 the s -1 ), the light / dark cycle is 12 hours, low temperature treatment is carried out, and the treatment is continuous for 2 days.

[0050] Extract the total RNA: Cut the mature leaves, add liquid nitrogen to quickly grind them into powder, transfer to a 2ml centrifuge tube, add 1.5ml TRE-Trizol (Guangzhou Boli Biotechnology Co., Ltd.) reagent, shake vigorously for 15 seconds, and ice-bath for 5 minutes . Centrifuge at 10000rpm for 5min, pipette the supernatant into a 2ml centrifuge tube, add 0.3ml chloroform, shake vigorously for 15 seconds, and place in ice bath for 10 minutes. Centrifuge at 12000rpm for 15 minutes, draw the upper aqueous phase (≤0.7ml) into a 1.5ml centrifuge tube, add 0.4ml isopropanol and 0.4ml Buffer A (1.2M NaCl, ...

Embodiment 2

[0066] The construction of embodiment 2MfEF2 plant expression vector pBI-MfEF2

[0067] Double enzyme digestion of plasmid pGM-MfEF2 and vector pBI121: double enzyme digestion of recombinant plasmid pGM-MfEF2 and pBI-121 expression plasmid with XbaI and BamHI, respectively. For enzyme digestion system and method, refer to the instructions of commercial enzymes from TaKaRa Company. Reaction system (50 μl): 20 μl pGM-MfEF2 (40ng / l), 2 μl 10× buffer, 2 μl restriction endonuclease XbaⅠ (10U / μl) and 24 μl sterile deionized water, mix well, and incubate at 37°C Cut for 2 hours; then add 2 μl of restriction enzyme BamHI (15 U / μL), and digest overnight at 30°C. After the reaction, incubate at 65°C for 15 minutes to inactivate the restriction endonuclease, and cool in an ice bath. The same method was used to perform double enzyme digestion on the binary expression plasmid pBI-121. The DNA gel recovery kit was used to purify and recover the double-digested MfEF2 fragment and the pBI1...

Embodiment 3

[0074] Embodiment 3MfEF2 gene is subjected to the expression situation of low temperature induction

[0075] 1. Obtaining the Medicago clover template under adverse conditions

[0076] Materials and treatments: Put the 8-10-week-old Medicago sativa plants into an artificial climate chamber at a temperature of 5°C (light intensity of 200 μmol m -2 the s -1 ), the light and dark time were both set to 12 hours of light. Continuous treatment for 4 days, low temperature treatment.

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

[0078] Synthesis of the first strand of cDNA: Using the above-prepared Medicago sativa RNA as a template, PrimeScript was used to TM RT reagent Kit (TaKaRa Company) ...

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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 a clover elongation factor 2MfEF2, its coding gene and its 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, resulting in reduced crop yields, and also affect the normal growth and quality of trees, fruit trees, flowers, and horticultural ornamental plants. The variety of plants is one of the main goals of the cultivation industry. However, plant stress tolerance is a quantitative trait involving at least hundreds of 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 translocase dependent on GTP hydrolysis, which mediates the translocation of ribosomes during protein synthesis. Every time a GTP is hydrolyzed, ribosomes will advance 3 base...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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