Paeonia suffruticosa PsDREB1 gene anti-drought and high salinity promoter and its expression and application

A promoter and anti-drought technology, applied in the field of plant genetic engineering research, can solve problems such as unclear mechanism of action

Inactive Publication Date: 2017-07-04
ZHEJIANG XIAOSHAN COTTON & FLAX RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its specific mechani...

Method used

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  • Paeonia suffruticosa PsDREB1 gene anti-drought and high salinity promoter and its expression and application
  • Paeonia suffruticosa PsDREB1 gene anti-drought and high salinity promoter and its expression and application
  • Paeonia suffruticosa PsDREB1 gene anti-drought and high salinity promoter and its expression and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of PsDREB1 promoter sequence DREBP in tree peony

[0028] In January 2016, the young leaves of the 3-year-old peony variety 'Luoyanghong' were taken as materials, and the total DNA was extracted using the Trizol kit produced by Omega Company, which was used as a template. According to the full-length sequence of PsDREB1 gene, design reverse primers SP1-CAACAGAAGGGGATCAGCGAAG (SEQ ID NO: 2), SP2-CTTTGGTTTTACCCTCTTGCTCGT (SEQ ID NO: 3), SP3-CGGATTTCTCATTTTCCCATTTC (SEQ ID NO: 4), using Genome Walking of TaKaRa Company Kit (Code No.6108), react with the above template. A product of about 2 kb in size was obtained by PCR amplification.

[0029] The above PCR product was recovered by using Takara’s Mini BEST Aragrose Gel DNA Extraction Kit Ver 4.0 (Code No. 9762) and named P1.

[0030] Using Solution I in TaKaRa's DNA Ligation Kit (Code No.6022), connect the P1 product to pMD 19-T Vector (Code No.6013), and thermally transform it into E.coli Competent Cel...

Embodiment 2

[0035] Example 2 Construction of recombinant vector pBI121-DREBP-GUS

[0036] Preparation of Insert DNA (target DNA): Using the promoter DREBP plasmid obtained above as a template, use the primer pair F2 (SEQ ID NO: 7) / R2 (SEQ ID NO: 8) using Takara’s PrimeSTAR ®® HS (Premix) (CodeNo. R040A) for PCR amplification, the amplification system is: DREBP plasmid (50-fold dilution) 1 μl, F2 (20 pmol / μl) 0.5 μl, R2 (20 pmol / μl) 0.5 μl, Prime STAR HS (Premix) 25 μl, add dH2O to 50 μl; reaction conditions: 98°C for 10s, 55°C for 15s, 72°C for 90s, a total of 30 cycles. Take 5 μl of the PCR product for agarose gel electrophoresis, and use Takara MiniBEST Agarose Gel DNA Extraction Kit Ver.4 .0 (Code No. 9762) to cut the gel to recover the target fragment, named Insert DNA.

[0037] Preparation of Vector DNA (carrier DNA): Using plasmid pBI121 as a template, use restriction endonucleases Hind III and XbaI for double enzyme digestion. The enzyme digestion reaction system is: pBI121 plasm...

Embodiment 3

[0039] Example 3 Genetic Transformation

[0040] The recombinant vector plasmid constructed above was transformed into Agrobacterium GV3101 by electroporation method, and then transformed into Arabidopsis thaliana by flower dipping method reported by Clough and Bent (1998). The F1 generation seeds of Arabidopsis transformed seedlings were collected, and the primary screening culture was carried out on 1 / 2 MS medium containing 40 mg / L kanamycin (Kan). The positive plants screened were then tested using the primer pair VP1 / VP2. For PCR identification, the plants identified as positive were continued to be planted to collect F2 generation seeds. Continue the above method to continue to obtain homozygous F3 generation seeds.

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Abstract

The invention discloses a paeonia suffruticosa PsDREB1 gene anti-drought and high salinity promoter and its expression and an application, which belong to the technical field of plant gene engineering, and clones a promoter DREBP of Paeonia suffruticosa PsDREB1 gene. A nucleotide sequence of the prmoter contains 2245 bp base, the promoter area contains three ABRE cis acting elements, which can specifically perform response reaction to ABA; and the promoter area contains a MYB binding site related to drought induction. An expression vector pBI121 DERBP GUS constructed by gene promoter fragment can be used for strongly inducing the expression of a target gene when the plants are suffered with adversity stress such as drought and high salinity.

Description

technical field [0001] The invention discloses a method for cloning, vector construction and function analysis of PsDREB1 promoter DERBP of tree peony, belonging to the field of plant genetic engineering research. Background technique [0002] peony( Paeonia suffruticosa ) is a traditional famous flower in my country. With the development of garden flower industry in our country, the beautification value and medicinal value of peony have been paid more and more attention. However, environmental factors have limited its cultivation and application. In order to grow graceful and luxurious peonies in Jiangnan and South China, it has become a very important and urgent task to cultivate peony varieties with strong stress resistance. In the early stage, we have obtained the PsDREB1 gene of peony anti-retroviral factor, and made it clear that this gene can improve the ability of plants to resist drought and high salinity. However, its specific mechanism of action has not been ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 刘慧春马广莹朱开元邹清成周江华史小华张加强
Owner ZHEJIANG XIAOSHAN COTTON & FLAX RES INST
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