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Agrobacterium tumefaciens mediated gene transformation method of botryosphaeria dothidea

A technology mediated by Botrytis and Agrobacterium, applied in the field of microorganisms, can solve the problem of not seeing gene transformation, and achieve the effect of ensuring the regeneration rate

Inactive Publication Date: 2014-07-30
ZHEJIANG UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

For Botrytis, a fungus with important economic significance, so far, we have not seen any reports on how to carry out gene transformation for this fungus

Method used

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  • Agrobacterium tumefaciens mediated gene transformation method of botryosphaeria dothidea
  • Agrobacterium tumefaciens mediated gene transformation method of botryosphaeria dothidea
  • Agrobacterium tumefaciens mediated gene transformation method of botryosphaeria dothidea

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Embodiment Construction

[0040] The method of the present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

[0041] 1. Preparation of Botrytis protoplasts

[0042] (1) Preparation of reagents and medium

[0043] ① 0.7M sodium chloride solution: Dissolve 40.95g sodium chloride (NaCl) in 1000mL water, and perform conventional high-temperature sterilization (1.1 atmospheres, 121°C for 20 minutes) after constant volume.

[0044] ②1M Tris-Cl, pH=7.5: Tris(hydroxymethyl)aminomethane (Tris(hydroxymethyl)aminomethane, generally abbreviated as Tris) 121.14 was dissolved in 600mL double-distilled water, adjusted to pH=7.5 with concentrated hydrochloric acid, and then adjusted to volume with double-distilled water to 1 L, perform conventional high-temperature sterilization (1.1 atmospheres, sterilize at 121°C for 20 minutes), and set aside.

[0045] ③Cell wall degrading enzyme solution: Weigh 100mg of Driselase and 100mg of Lysing Enzyme on a ba...

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Abstract

The invention discloses an agrobacterium tumefaciens mediated gene transformation method of botryosphaeria dothidea. The method comprises the steps of preparing the protoplast of botryosphaeria dothidea, and carrying out renewal culture on the protoplast; transforming a plasmid vector containing a target gene into agrobacterium tumefaciens; mixing agrobacterium tumefaciens with the protoplast, co-culturing agrobacterium tumefaciens and the protoplast on an induction plate, and then transferring agrobacterium tumefaciens and the protoplast to a screening plate to be cultured, thus obtaining a potential transformant; confirming whether a potential transformant genome contains the target gene, thus obtaining a genetically stable transformant. The method has the beneficial effects that the protoplast is used as a transformation material and the target gene is transferred into botryosphaeria dothidea by utilizing agrobacterium tumefaciens mediation and is integrated into the genome; before co-culture, the protoplast needs renewal culture; when the renewal culture time is 2-6 hours, transformation materials in each tube can obtain 20-30 potential transformants; when the renewal culture time is more than 6 hours or less than 2 hours, the transformation efficiency is quickly reduced, and only few potential transformants can be obtained and even the potential transformants are difficult to obtain.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to an Agrobacterium-mediated gene transformation method for Botrytis spp. 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 iso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/21C12R1/645
Inventor 王洪凯林福呈
Owner ZHEJIANG UNIV
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