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Cloning of Calycanthus praecox WRKY transcription factor gene CpWRKY71 and its promoter and application thereof

A technology of transcription factors and promoters, applied in the field of genetic engineering, can solve the problems of gene cloning and functional research of the undiscovered Wintersweet WRKY transcription factor

Inactive Publication Date: 2019-09-03
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the cloning and functional study of the WRKY transcription factor gene

Method used

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  • Cloning of Calycanthus praecox WRKY transcription factor gene CpWRKY71 and its promoter and application thereof
  • Cloning of Calycanthus praecox WRKY transcription factor gene CpWRKY71 and its promoter and application thereof
  • Cloning of Calycanthus praecox WRKY transcription factor gene CpWRKY71 and its promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Isolation of Wintersweet CpWRKY71 Gene

[0036] According to the known sequence fragments in the wintersweet flower transcriptome database, the software Primer primer 5.0 was used to design specific primers for PCR amplification. The primer sequences are as follows:

[0037] CpWRKY71-F: 5'-aagctaaacctcttccctct-3' (SEQ ID No.4)

[0038] CpWRKY71-R: 5'-ccaactaggatgttggttc-3' (SEQ ID No.5)

[0039] Using Wintersweet cDNA as a template, the CpWRKY71 gene of Wintersweet was amplified. The PCR reaction system was as follows: 10×Taq PCRBuffer 2.5 μL, dNTP (10 mM) 1.5 μL, CpWRKY71-F (10 μM) 1 μL, CpWRKY71-R (10 μM) 1 μL, TaKaRaEx TaqTM 0.2μL, template 2μL, add ddH 2 0 to 25 μL. The reaction conditions are as follows: 95°C for 5 min; 95°C for 30s, 52°C for 30s, 26 cycles; 72°C for 1 min; 72°C for 10 min.

[0040] After the PCR product was recovered, it was connected to the T vector, transformed into Escherichia coli competent cells, and the recombinants were picked...

Embodiment 2

[0042] Example 2 Analysis of the expression characteristics of Wintersweet CpWRKY71 gene

[0043] 1 RNA extraction

[0044] (1) The equipment used for RNA extraction, mortar, mortar stick, spoon, scissors, etc., were wrapped in tin foil, placed in an oven at 180°C for 4 hours, and cooled for later use.

[0045] (2) Take wintersweet leaves, grind them fully with liquid nitrogen in a mortar, quickly pour them into a RNase-removing centrifuge tube, add 600 μL Trizol (plant RNA extraction reagent, Thermo, USA), vortex to mix, and store at room temperature Leave it for 10min.

[0046](3) Add 120 μL of chloroform, vortex and mix well, and let stand at room temperature for 10 min.

[0047] (4) Centrifuge at 12,000 rpm for 10 min at 4°C, and pipette 200 μL of the supernatant into another centrifuge tube.

[0048] (5) Add 2 times the volume of absolute ethanol and mix well, then centrifuge at 12000rpm at 4°C for 30min, discard the supernatant, and keep the precipitate.

[0049] (6)...

Embodiment 3

[0065] Cloning of Example 3 Wintersweet CpWRKY71 promoter

[0066] 1 Acquisition of the promoter fragment of CpWRKY71 gene of Wintersweet

[0067] The promoter of CpWRKY71 gene was amplified with Clontech Chromosome Walking Kit. Based on the sequence of the cloned Wintersweet CpWRKY71 gene, use the software Primer primer 5.0 to design specific primers for PCR amplification. The primer sequences are as follows:

[0068] pCpWRKY71-GSP1: 5'-gccaacactccagaagcactccc-3' (SEQ ID No. 12)

[0069] pCpWRKY71-GSP2: 5'-catcgccagagccgaacacg-3' (SEQ ID No. 13)

[0070] According to the steps of the Chromosome Walking Kit, the promoter of the CpWRKY71 gene of Wintersweet is amplified, and the PCR reaction system and reaction conditions are as follows:

[0071] First round: 10×Advantage 2PCR Buffer 2.5μL, dNTP (10mM) 0.5μL, AP1 (provided in the kit) (10mM) 0.5μL, GSP1 (10mM) 0.5μL, Advantage 2 Polymerase Mix (50×) 0.5μL, template 2μL , plus ddH 2 O to 20 μL; 94°C for 25s, 72°C for 3min, ...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to the cloning of a Calycanthus praecox WRKY transcription factor gene CpWRKY71 and its promoter and an application thereof. The object of the present invention is to provide a new option for the study of adversity stress of Calycanthus praecox. The present invention provides the Calycanthus praecox WRKYtranscription factor gene CpWRKY71, and its encoded protein has an amino acid sequence shown as SEQ ID No.3. The Calycanthus praecox CpWRKY71 gene is cloned for the first time, and the expression characteristics of CpWRKY71 gene are detected by a real-time fluorescent quantitative PCR technology, and the function of the gene promoter is verified in Arabidopis thaliana by a transgenic technology.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the cloning and application of the wintersweet WRKY transcription factor gene CpWRKY71 and its promoter. Background technique [0002] Wintersweet (Chimonanthus praecox) is a deciduous shrub of the family Chimonanthus genus Chimonanthus praecox, native to China and widely distributed in south-central and southwest China. Wintersweet is a rare winter flowering plant, and the flowering period is from December to February of the following year. With its unique flower color and pleasant fragrance, wintersweet can be used as potted plants, ornamental cut flowers and landscaping plants. At present, some progress has been made in the research of wintersweet on abiotic stress and flower development. [0003] WRKY transcription factor is a kind of plant-specific superfamily transcription factor, which is named because the N-terminus of the WRKY domain contains a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/113C07K14/415A01H5/02
CPCC07K14/415C12N15/8271C12N15/8273C12N15/8291C12N15/8293C12N15/8297
Inventor 黄仁维刘道凤眭顺照马婧李志能李名扬
Owner SOUTHWEST UNIVERSITY
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