Gene CdERF1 of Cynodon dactylon significantly induced by low temperature and application thereof

A low-temperature induction, bermudagrass technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of weak research on the molecular mechanism of bermudagrass cold resistance, unclear genetic background of bermudagrass, lack of original cold resistance genes, etc.

Active Publication Date: 2018-11-02
武汉植物园园艺中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But at the same time, because the genetic background of bermudagrass is still unclear, relevant research on it at the level of molecular biology is rel

Method used

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  • Gene CdERF1 of Cynodon dactylon significantly induced by low temperature and application thereof
  • Gene CdERF1 of Cynodon dactylon significantly induced by low temperature and application thereof
  • Gene CdERF1 of Cynodon dactylon significantly induced by low temperature and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0058] 2. Example 2: Molecular Cloning of Bermudagrass CdERF1 Gene

[0059] Sequencing found that the 5' end of the ERF2 sequence started from the start codon ATG, followed by 3'RACE PCR amplification; according to TaKaRa 3'-Full RACE Core Set with PrimeScript TM According to the requirements of the RTase manual, 3 specific primers were designed for 3’-RACE, and the primers are as follows:

[0060] ERF2-3'RACEouter, 5'-CTCGTGGATTCTCCAAATGGTGCA-3';

[0061] ERF2-3'RACEinner1, 5'-AGGACGAGAGAACCCAGTATTACC-3';

[0062] ERF2-3'RACEinner2,5'-ACCAAGCGTAGAAAACGGCACAGA-3';

[0063] The principle of 3'-RACE is: use Total RNA as a template, use 3'RACE Adapter primers, perform reverse transcription under the action of reverse transcriptase PrimeScript Reverse Transcriptase, and synthesize the first standard cDNA; use the upstream outer specific primer ( ERF2-3'RACEouter) and 3'RACE OuterPrimer for the first round of PCR amplification; if the first round of PCR reaction fails to obtain ...

Embodiment 3

[0068] 3. Example 3: Analysis of the expression profile of the transcription factor gene CdERF1 under stress

[0069] ERF transcription factors respond to a wide range of biotic and abiotic stresses, such as low temperature, drought, high salinity, pests and mechanical damage, etc. can induce the expression of ERF transcription factor genes, and different ERF transcription factors show different behaviors under different stress conditions. expression pattern; the primer sequences used in this experiment are as follows:

[0070] CdERF1-q up: 5'-ACTGCAGAGGTACACCT-3';

[0071] CdERF1-q dn: 5'-GAAGAGCCACCACCATT-3';

[0072] CdACT2up: 5'-TCTGAAGGGTAAGTAGAGTAG-3';

[0073] CdACT2dn: 5'-ACTCAGCACATTCCAGCAGAT-3'.

[0074] Mature bermudagrass grown for 2 months were given low temperature (4°C), drought (25% PEG), high salt (200 mM NaCl), abscisic acid (100 μM ABA), ethylene (100 μM ACC) and jasmonic acid (100 μM JA), etc. Different treatments; the leaves of the seedlings were cut a...

Embodiment 6

[0120] 6. Embodiment 6: Cultivation and genetic transformation of Arabidopsis thaliana

[0121] 1) Culture of Arabidopsis

[0122] A. Put a little Arabidopsis seeds into a centrifuge tube, add sodium hypochlorite to treat for 5-6 minutes, absorb the sodium hypochlorite and wash the seeds with sterile water 4-6 times;

[0123] B. Spread the seeds on the surface of MS solid medium, and disperse the seeds evenly; place the culture dish at 4°C for 3 days, then transfer it to the Arabidopsis room (22°C) for germination and culture for 10 days, and then transplant ;

[0124] C, plant about 6-9 Arabidopsis thaliana in the sterilized nutrient soil of every pot;

[0125] D. When Arabidopsis thaliana is bolted, the main stem is cut off from the base, so as to promote the growth of side branches and bolting and flowering, and then be used for transformation;

[0126] 2) Transfection of Arabidopsis thaliana by soaking flowers

[0127] A, select Arabidopsis thaliana with more flowering...

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Abstract

The invention discloses gene CdERF1 of Cynodon dactylon significantly induced by low temperature and application thereof and belongs to the field of plant gene engineering. The gene CdERF1 has an ORF(open reading frame) sequence shown as in SEQ ID NO. 1 and an encoded amino acid sequence shown as in SEQ ID NO. 2. Application of the gene CdERF1 refers to: inferring that the gene CdERF1 may involvein plant stress resistance by analyzing different stress treatment expression modes of the gene CdERF1 in Cynodon dactylon; B, converting to Arabidopsis thaliana by Agrobacterium-mediated transformation, and studying the anti-stress function of the gene CdERF1 in transgenic Arabidopsis thaliana so that basis is laid for the application of the gene CdERF1 in other plants; C, applying the gene CdERF1 to plant transformation to improve plant cold tolerance. The gene CdERF1 herein is a transcription factor gene that may specifically regulate cold resistance improvement for plants; the gene CdERF1is originated from plants and causes low ambient influence; transgenic Arabidopsis thaliana having high tolerance to low temperature stress is attained.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to a gene CdERF1 significantly induced by low temperature in bermudagrass and its application; Sex-related novel gene CdERF1. Background technique [0002] Low temperature is one of the important environmental factors affecting plant growth and development. It limits the geographical distribution of plants and determines the growth cycle of crops. Low temperature stress will lead to a series of changes in plants, including changes in cell membrane stability, antioxidant enzyme activities, and osmotic adjustment substances. At the same time, in order to better resist these stresses, complex and efficient regulatory pathways for stress signal responses have evolved in plants. The stimulating signal acquired by this complex signal transduction network will activate the expression of transcription factors, through the combination of transcription factors and cis-acting element...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8273
Inventor 陈良户正荣谢燕
Owner 武汉植物园园艺中心有限公司
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