Promoter of watermelon ClCP2 gene and preparation method and application thereof

A technology of promoter and promoter sequence, applied in the field of plant genetic engineering, can solve the problem of no watermelon endogenous promoter, etc., and achieve the effects of stable and reliable results, high expression intensity and easy detection.

Inactive Publication Date: 2016-03-23
WUHAN INST OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

There is no corresponding watermelon endogenous promoter

Method used

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  • Promoter of watermelon ClCP2 gene and preparation method and application thereof
  • Promoter of watermelon ClCP2 gene and preparation method and application thereof
  • Promoter of watermelon ClCP2 gene and preparation method and application thereof

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Experimental program
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preparation example Construction

[0039] A preparation method of the promoter of the above-mentioned watermelon ClCP2 gene, it comprises the steps:

[0040] Step 1: Design a pair of primers for PCR amplification based on the sequence of about 2 kb upstream of the 5' end of the ClCP2 gene obtained by sequencing the whole watermelon genome, and obtain the promoter sequence of the 2 kb upstream of the 5' end of the watermelon ClCP2 gene. The primers used are:

[0041] pClCP2FP: 5'-(KpnI) CGGGGTACC AAGAGACGACGAATGCCAATA-3';

[0042] pClCP2RP:5′-(XbaI) CTAGTCTAGA TTTCACCGCTAAGAATCGAAG-3';

[0043] Step 2: The primer pClCP2FP used in the gene 5' end upstream 2kb promoter sequence design of the ClCP2 obtained according to the watermelon whole genome sequencing: 5'-(KpnI) CGGGGTACC AAGAGACGACGAATGCCAATA-3'; and pClCP2RP:5'-(XbaI) CTAGTCTAGA TTTCACCGCTAAGAATCGAAG-3′, using the CTAB method to extract the genomic DNA of watermelon leaves, using the extracted DNA as a template for polymerase chain reaction amplifica...

Embodiment 1

[0052] Embodiment 1: the promoter preparation of watermelon ClCP2 gene

[0053] 1. Preparation of watermelon genomic DNA

[0054] Utilize CTAB method (J. Sambrook.D.W.Russell is written, and Huang Peitang etc. translates molecular cloning test guideline (third edition) Science Press, hereinafter the same) to extract watermelon leaf genomic DNA, after agarose gel electrophoresis detection, in Store at -20°C.

[0055] 2. Promoter cloning and sequence analysis of watermelon ClCP2 gene

[0056] Carry out PCR amplification using watermelon genomic DNA as a template, the reaction system is 50 μl, add 5 μl of 10×ExTaqbuffer, 4 μl of dNTP, 1 μl of 5’ primer, 1 μl of 3’ primer, 0.5 μl of ExTaq, 1 μl of DNA template, about 100 ng, ddH 2 O 37.5 μl. According to the upstream 2kb sequence of the ClCP2 gene obtained by sequencing the whole watermelon genome, the primers used for PCR design are pClCP2FP: 5'-(KpnI) CGGGGTACC AAGAGACGACGAATGCCAATA-3' and pClCP2RP:5'-(XbaI) CTAGTCTAGATTTC...

Embodiment 2

[0058] Embodiment 2 Construction of watermelon ClCP2 promoter recombinant expression vector and transformation of Agrobacterium tumefaciens

[0059] (1) Digest pMD18-pClCP2 and pBI121 plasmids with XbaI / KpnI respectively (purchased from TaKaRa Company, Chen, P.Y., Wang, C.K., Soong, S.C.To, K.Y. Complete sequence of the binary vector pBI121 and its application incloning T-DNA insertion from transgenic plants. Mol. Breed-129, 287 below same), the enzyme digestion system is 10 μl. The pClCP2 fragments and pBI121 fragments excised by the enzyme were recovered from the gel, and after being detected correctly by 1.0% mass-volume ratio agarose gel electrophoresis, they were ligated at 16°C, and transformed into E. Screening on the solid LB culture medium plate of L), pick white bacterial spot and do colony PCR, extract plasmid, the recombinant vector of correct size (enzyme cutting small fragment is 2010bp) is named as pBI-pClCP2 ( image 3 ).

[0060] (2) Use the freeze-thaw meth...

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Abstract

The invention discloses a preparation method and application of a promoter of a watermelon ClCP2 gene. The preparation method comprises the steps that 1, PCR amplification is performed according to a pair of upstream sequence design primers which are in the size about 2 kb and are obtained through watermelon whole genome sequencing to amplify an upstream promoter sequence of the watermelon ClCP2 gene 5'; 2, watermelon leaf genome DNA is extracted through a CTAB method according to the upstream sequence design primers which are in the size about 2 kb and are obtained through watermelon whole genome sequencing, PCR amplification is performed by taking the watermelon leaf genome DNA as a template, after a gel extraction kit performs purification and recovery, a PCR product is connected to a pMD18-T carrier, competent cells of a escherichia coli DH5alpha strain are converted through a thermal stimulation method, the converted competent cells are picked for positive clone, PCR detection on the positive clone and enzyme cutting verification are performed, sequencing is performed, and then an upstream 2014-bp flanking sequence of the ClCP2 gene is obtained. The promoter has the good application potential on the aspects such as watermelon transgenosis safety.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a watermelon ClCP2 (Cla016594) gene promoter, its preparation method and application. Background technique [0002] The promoter of a plant gene is a DNA sequence located in the upstream region of the 5' end of the gene and contains cis-acting elements, which determines the specificity, direction and efficiency of downstream gene transcription, and is the most critical factor in the gene transcription regulation mechanism and expression mode . In addition, the expression of exogenous genes in plant cells is the key to plant genetic engineering research, and the expression of exogenous genes first depends on the initiation of their transcription. To some extent, the promoter determines the temporal and spatial order of gene expression and the expression intensity . Therefore, it is of great scientific significance to discover and study new promoters for the stu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82C12N15/66A01H5/02A01H5/06
CPCC07K14/415C12N15/66C12N15/8203C12N15/8225C12N15/8226C12N15/8227
Inventor 阳永学祝菊红杨洁赵志远李其友张安华王萍
Owner WUHAN INST OF AGRI SCI
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