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Plasmid vector, building method thereof and application

A technology of plasmid vector and botrytis, which is applied in the field of microorganisms, can solve the problems of low transformation efficiency and achieve the effect of improving transformation efficiency

Active Publication Date: 2017-09-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention provides a plasmid vector, which solves the problem of low transformation efficiency when the existing plasmid is transformed into Botrytis

Method used

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  • Plasmid vector, building method thereof and application
  • Plasmid vector, building method thereof and application
  • Plasmid vector, building method thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of the plasmid vector pBDH3G-Thy for efficient transformation of Botrytis

[0055] Plasmid pEGFP of the present invention was purchased from CLONTECH Company; information on plasmid pCAMBIA1300: Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation.Plant Mol Biol 25:989-994.; Information on plasmid pCB1003 : Carroll AM, Sweigard JA, Barbara VC (1994) Improved vectors for selecting resistance to hygromycin. Fungal Genetic News, 41:22. The above three plasmids are all kept in the Fungal Biology Laboratory of Agricultural College of Zhejiang University.

[0056] In the Botrytis genome database ( http: / / genome.jgi.doe.gov / Botdo1 / Botdo1.home.html ) to find the histone H3 gene and the transcription elongation factor α (TEF) gene of Botrytis aureus, respectively download about 1500 bp upstream (containing the promoters of the respective genes), and then design primers. Then use...

Embodiment 2

[0077] Example 2 Preparation of Botrytis botrytis protoplasts

[0078] 1. Preparation of reagents and medium

[0079] (1) 0.7M sodium chloride solution: 40.95g of sodium chloride (NaCl) was dissolved in 1000mL of water, and then subjected to conventional high-temperature sterilization (1.1 atmospheres, 121°C for 20 minutes).

[0080] (2) 1M Tris-Cl, pH=7.5: Tris(hydroxymethyl)aminomethane (Tris(hydroxymethyl)aminomethane, generally abbreviated as Tris) 121.14 was dissolved in 600mL redistilled water, adjusted to pH=7.5 with concentrated hydrochloric acid, and then redistilled Dilute the water to 1 L, perform conventional high-temperature sterilization (1.1 atmospheres, sterilize at 121°C for 20 minutes), and set aside.

[0081] (3) Cell wall degrading enzyme solution: Weigh 100 mg of Driselase and 100 mg of Lysing Enzyme on a balance, dissolve them in 10 mL of 0.7M sodium chloride solution, and filter them with a diameter of 0.22 μm Sterilized by membrane filtration, ready to ...

Embodiment 3

[0094] Example 3 Agrobacterium-mediated botrytis DNA transformation

[0095] 1. Preparation of Botrytis strain FY-31:

[0096] Inoculate the PDA plate with the Botrytis strain FY-31 preserved in our laboratory, culture it at 25°C for 5 days, and set it aside.

[0097] 2. Plasmid preparation

[0098] The pBDH3G-Thy1 and pBDH3G-Thy2 in Example 1, and the PKO1-HPH plasmid used for the control were extracted, and the concentration of the plasmid was adjusted to 0.25 μg / μL.

[0099] 3. Transform the plasmid vector into Agrobacterium by freeze-thaw method

[0100] (1) The formula of LB solid medium is as follows: tryptone (Trypone) 10g, yeast extract (Yeast extract) 5g, sodium chloride (NaCl) 10g, agar 15g, water 1000mL, pH=7.0. Routine high-temperature sterilization (1.1 atmospheres, 121°C for 20 minutes).

[0101] LB liquid medium: Agar is not added to LB solid medium, and the others are the same.

[0102] (2) Agrobacterium strain AGL1 was streaked and activated on LB plates,...

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Abstract

The invention discloses a plasmid vector, a building method thereof and an application. PCAMBIA1300 is modified to form the plasmid vector, botryosphaeria dothidea histone H3 gene promoters, botryosphaeria dothidea transcription elongation factor alpha gene promoters and resistance genes are inserted into pCAMBIA1300 multiple cloning sites, and the resistance genes are positioned at downstream positions of the botryosphaeria dothidea transcription elongation factor alpha gene promoters. According to the plasmid vector, segments among botryosphaeria dothidea histone H3 genes and the transcription elongation factor alpha gene promoters are selected, and genes connected at the back can be efficiently started, so that conversion efficiency of the plasmid vector is greatly improved. According to the building method, the botryosphaeria dothidea is efficiently converted by the aid of exogenous genes mediated by agrobacterium tumefaciens, and a feasible scheme is provided for research and development of aspects such as genetic modification of the botryosphaeria dothidea, development of interested genes, drug target genes separating pathogenic fungi and development of gene functions of the botryosphaeria dothidea.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a plasmid vector and its construction method and application. Background technique [0002] Botryosphaeria dothidea is a common and important fungus with global distribution and has important development and utilization value. Some of these fungi can saprophyte on a variety of decaying wood, some parasitize on a variety of woody plants, causing diseases of woody plants, and some form symbionts with plants, which are endophytic fungi of many plants. Botrytis, which lives saprophytically on rotting wood, can decompose woody plant residues and plays an important role in the material cycle and energy cycle of the ecosystem; at the same time, this type of fungus can also be developed to produce lignin enzymes and Cellulase has good prospects for economic development. The strains belonging to endophytic fungi can be used as bioreactors. For example, a fungus isolat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/66C12N1/15C12R1/645
CPCC12N15/80C12N2800/10C12N2830/001C12N1/145C12R2001/645
Inventor 王洪凯林福呈
Owner ZHEJIANG UNIV
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