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Plant expression vector of Tanba black soybean C2H2 zinc finger protein gene STOP1 and application thereof

A plant expression vector and zinc finger protein technology, applied in the field of plant genetic engineering, to achieve the effect of enhancing the secretion of malic acid and citric acid

Inactive Publication Date: 2013-03-06
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has been clearly verified using mutant technology STOP1 Function in plant resistance to aluminum, but the application of overexpressing the gene in plants and improving the resistance of transgenic plants to aluminum toxicity has not been reported

Method used

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  • Plant expression vector of Tanba black soybean C2H2 zinc finger protein gene STOP1 and application thereof
  • Plant expression vector of Tanba black soybean C2H2 zinc finger protein gene STOP1 and application thereof
  • Plant expression vector of Tanba black soybean C2H2 zinc finger protein gene STOP1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: STOP1 Gene cDNA amplification, TA cloning and sequence analysis

[0035] According to NCBI GmSTOP1 The published ORF sequence, the designed primers are:

[0036] Upstream primer: 5′- GGTACC ATGTTGAATAACCTTTCCTTCC-3′

[0037] Downstream primer: 5′- GGTACC GATAGCGACAATTTATAA-3′

[0038] Add the GGTACC characteristic sequence to the 5' end and 3' end of the upstream primer, and thus form Kpn The I enzyme cutting site is amplified with the first-strand cDNA of Danbo black soybean as a template to obtain STOP1 The full-length cDNA of the gene;

[0039] After aluminum-resistant soybean variety Danba black soybeans were treated with aluminum for 24 hours, cut 0.1g of roots, grind them with liquid nitrogen, add 1ml of TRIzoL extract, let stand at room temperature for 5min, then transfer to a centrifuge tube, then add 0.2ml of chloroform, shake and mix well , centrifuge for 15min (12000rpm), transfer the supernatant to a new tube, add 0.5ml of isopropano...

Embodiment 2

[0042] Example 2: Construction of plant expression vector pPZP-35S- STOP1

[0043] use KpnI pMD18- STOP1 and pPZP-221 vector ( Figure 5 A), the excised vector and insert fragments were separated by agarose gel electrophoresis, and pMD18- STOP1 produced after being cut STOP1 The gene fragment (about 1.2kb) and the linear vector fragment pPZP-221 generated after pPZP-221 were cut, and then pPZP-221 and STOP1 The DNA fragment of the gene produces the plant expression vector pPZP-35S- STOP1 ( Figure 4 ). Conversion of high efficiency (10 8 ) Escherichia coli competent cells (DH5α, purchased from Tiangen Biochemical Technology Co., Ltd.), spread the transformed Escherichia coli on a plate added with spectinomycin (Spe, 50 μg / ml), cultivate overnight at 37 °C, and screen Spe-resistant recombinant colonies. Plasmids were extracted from Spe-resistant recombinant colonies for PCR detection ( Figure 5 B). use KpnI Enzyme digestion detection of pPZP-35S- STOP1 , two ...

Embodiment 3

[0044] Embodiment 3: use STOP1 Agrobacterium

[0045] Competent cells of Agrobacterium were prepared, and the above-constructed plant expression vector pPZP-35S- STOP1 Transform into Agrobacterium (C58Cl(pPMP90)), and select transformants on plates added with spectinomycin. Take a small amount of plasmid and add it to the competent cells of Agrobacterium, mix gently; add the mixture into the pre-cooled electroporation cup, tap the cup body gently to make the mixture fall to the bottom of the cup; place the electroporation cup on the electroporation In the chute of the BIO-RAD instrument, use a 1mm electric shock cup and 200 ohm, 2.5kV / 0.2cm parameters for electric shock, take out the electric conversion cup immediately after the electric shock, quickly add 0.5 ml SOC medium, mix well, and transfer to In a 1.5ml centrifuge tube; 28°C, 200rpm shaker culture for 3-5h; room temperature, 7500rpm centrifuge 1min, discard most of the supernatant, retain 100μl to suspend the cells; ...

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Abstract

The invention discloses a plant expression vector Ppzp-35S-STOP1 of Tanba black soybean C2H2 zinc finger protein gene STOP1 and an application thereof for improving plant aluminum toxicity stress resistance. The vector is the plant expression vector containing constitutive promoter (35S) of cauliflower mosaic virus and C2H2 zinc finger protein gene STOP1 induced by the Tanba black soybean aluminum, the STOP1 gene is amplified from tanba black soybean roots, and the over-expression of the STOP1 gene in tobacco is controlled by utilizing the constitutive promoter, so that the aluminum toxicity resistance of the tobacco can be improved. The STOP1 transgene tobacco forcedly grows in solution containing 30microliters aluminum trioxide (AlCl3) for 24hours, the malic acid and the citric acid secreted from roots of the transgene tobacco as well as the aluminum resistance can be remarkably higher than that of a wild contrast plant, and the aluminum content on the root of the tobacco is remarkably lower than that of the wild plant.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to a C2H2 type zinc finger protein gene induced by aluminum in Danba black soybean roots STOP1 The plant expression vector pPZP-35S- STOP1 and its application in improving the ability of plants to resist aluminum toxicity stress. Background technique [0002] Aluminum toxicity is a major limiting factor for plant growth and crop yield in acidic soils. Acidic soils account for 30% of the world's arable soils, and are mainly distributed in South Africa, Central Asia, and Southeast Asia, developing countries with the greatest food demand. The acidic soil in my country is mainly distributed in the tropical, subtropical and Yunnan-Guizhou-Sichuan areas south of the Yangtze River, covering an area of ​​2.04 million hectares. The pH value of most of the soil is less than 5.5, and a large part of it is less than 5.0. In agriculture, the application of quicklime is usually used to...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
Inventor 陈丽梅陈奇武孔焕李昆志玉永雄
Owner KUNMING UNIV OF SCI & TECH
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