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Plant expression vector of broad bean VFPPIC gene and application of plant expression vector

A plant expression vector, PK-35S-VFPP1C technology, used in applications, plant products, genetic engineering, etc.

Inactive Publication Date: 2015-07-01
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on whether PP1c is involved in the response to formaldehyde stress

Method used

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  • Plant expression vector of broad bean VFPPIC gene and application of plant expression vector
  • Plant expression vector of broad bean VFPPIC gene and application of plant expression vector
  • Plant expression vector of broad bean VFPPIC gene and application of plant expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Reality Embodiment 1: under formaldehyde stress VFPP1C Gene expression level detection

[0044] Robust broad bean plants with consistent growth for 4 weeks were selected in the greenhouse. During the experiment, the flowerpots planted with broad beans were covered with plastic bags to prevent the soil and microorganisms in the soil from absorbing formaldehyde, and then the broad bean plants were placed in the air containing 0, 5, 10 , 20 and 40ppm gas formaldehyde in the glass airtight chamber, the device size is 700 mm × 600 mm × 700 mm (length × width × height), no gas leakage has been detected. Light sources are provided on both sides of the sealed chamber, and the light intensity in the chamber is 600 μmol / m 2 ·s, four small fans are installed at the corners of the sealed chamber to accelerate the circulation of gas formaldehyde in the chamber and evenly distribute it in the chamber space. The temperature and humidity in the sealed chamber are determined by the ...

Embodiment 2

[0045] Example 2: Plant expression vector PK-35S- VFPP1C build

[0046] 1. External source VFPP1C Gene acquisition and TA cloning

[0047] Strategies for constructing plant expression vectors such as figure 2 As indicated, total RNA was extracted from broad bean leaves ( image 3 A), after reverse transcription into cDNA, using cDNA as a template, amplified with specific primers with restriction enzyme sites BamHI and XhoI VFPP1C CDS fragment (972bp) ( image 3 B). Upstream primer: 5'- GGATCC ATGAGTGCACAAGGACAAC-3' (contains BamH I restriction site), downstream primer: 5'- CTCGAG TCACATCTTGTTTGACATCAC-3' (contains XhoI restriction site). recovery and purification VFPP1C Gene fragment( image 3 C), and connect it to pMD18T (Dalian Bao Biological Company) vector, transform Escherichia coli competent DH5α (Tiangen Biochemical Technology), and use alkaline lysis to extract plasmid DNA ( Figure 4 A), after 1% agarose gel electrophoresis, select the recombinant plas...

Embodiment 3

[0052] Embodiment 3: plant expression vector PK-35S- VFPP1C Screening of Transformed Agrobacterium and Transgenic Tobacco

[0053] Take a small amount of detected correct plant expression vector PK-35S- VFPP1C Add the plasmid into the competent cells of Agrobacterium, mix gently; add the mixture into the cold electroporation cup, tap the body of the cup gently to make the mixture reach the bottom of the cup; place the electroporation cup in the chute of the electrotransformer, Use 200 ohm, 2.5kV / 0.2cm parameters for electric shock. After electric shock, immediately take out the electric cup and quickly add LB medium, mix well, transfer to a 1.5mL centrifuge tube, and incubate on a shaker at 28°C and 200rpm for 3-5h; room temperature Centrifuge at 7500rpm for 1min, discard the supernatant to 100μL to suspend the cells; spread the bacteria on LB solid medium containing spectinomycin (Spe) antibiotics and culture at 28°C for 2 days; select positive clones and perform bacteri...

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PUM

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Abstract

The invention discloses a plant expression vector of a broad bean VFPPIC gene and an application of the plant expression vector. Through the invention, the condition that the expression level of the VFPPIC gene in broad bean is significantly inhibited under the formaldehyde stress is found out for the first time; a CDS sequence of the VFPPIC gene is obtained by cloning from broad bean leaves; a plant expression carrier PK-35S-VFPPIC is constructed through a Gateway technique; a wild tobacco is transformed by agrobacterium-mediated leaf-disk transformation method; a transgenic tobacco capable of correctly expressing the VFPPIC gene is obtained by screening; and finally, analysis of the formaldehyde absorbing ability is carried out on the obtained transgenic tobacco. An experiment result shows that the formaldehyde absorbing ability of the transgenic tobacco can be significantly improved by over-expression of an exogenous VFPPIC gene in the tobacco.

Description

technical field [0001] The present invention relates to a kind of broad bean VFPP1C The plant expression carrier of the gene and its application in improving the formaldehyde absorption capacity of plants belong to the field of plant molecular genetic engineering. Background technique [0002] Formaldehyde is widely used in industrial production and is the most widely used chemical raw material in the adhesive industry. With the development of the economy and the improvement of people's living standards, building decoration materials made of various raw materials have entered various indoor public places and homes, making formaldehyde the most representative chemical substance recognized as indoor air pollution. The survey results of newly renovated houses show that the concentration of formaldehyde in the indoor air is 6.65 times that of the outdoor air, and the concentration of formaldehyde exceeds the standard by 100 times when the doors and windows are closed. Formalde...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/55A01H5/00
Inventor 陈丽梅周升恩
Owner KUNMING UNIV OF SCI & TECH
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