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Genetic transformation methods of fingerlike blue mold mediated by agrobacterium tumefaciens

A genetic transformation method, the technology of Agrobacterium tumefaciens, is applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials. It can solve the problems of difficult preparation of protoplasts and unstable transformants, and achieve Stable transformants, high regeneration rate, simple effect requirements

Inactive Publication Date: 2008-05-21
ZHEJIANG UNIV
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Problems solved by technology

[0014] The receptors used in the transformation method disclosed in this patent are protoplasts or mycelium. On the one hand, it is difficult to prepare protoplasts, and the separation of mycelia due to their properties may easily cause the instability of transformants.

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  • Genetic transformation methods of fingerlike blue mold mediated by agrobacterium tumefaciens

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Embodiment Construction

[0031] Medium formula:

[0032] MM medium: 10mM K 2 HPO 4 , 10mM KH 2 PO 4 , 4mM (NH 4 ) 2 SO 4 , 2.5mM NaCl, 2mM Mg SO 4 ·7H 2 O, 0.45 mM CaCl 2 2H 2 O, 9 μM FeSO 4 , 10 mM glucose, pH=6.9.

[0033] IM medium: 10mM K 2 HPO 4 , 10mM KH 2 PO 4 , 4mM (NH 4 ) 2 SO 4 , 2.5mM NaCl, 2mM MgSO 4 ·7H 2 O, 0.45 mM CaCl 2 2H 2 O, 9 μM FeSO 4 , 10 mM glucose, 40 mM 2-(N-morpholino)ethanesulfonic acid (MES), 0.5% glycerol, pH=5.3-5.5. Isolation and Purification of Penicillium digitatum

[0034] Pick moldy oranges, wipe the rotten parts with alcohol, cut off small pieces of tissue at the edge of the rotten parts with a sterilized blade, cut off three pieces of tissue from each rotten part, and place the three pieces of rotten tissues in a triangular direction. Put it on potato medium (PDA) (ampicillin 100 μg / ml, streptomycin 100 μg / ml to kill bacteria), culture at 25°C for 3-4 days, pick mycelium pieces from the edge of uncontaminated plaques and inoculate them on n...

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Abstract

The invention discloses a genetic transformation method of the agrobacterium tumefaciems mediation of penicillium digitatum; the method comprises the following steps that the plasmid is transferred into agrobacterium tumefaciems with the method of thermal stimulation to produce the agrobacterium tumefaciems which contains vectors; the agrobacterium tumefaciems which contains vectors is mixed with penicillium digitatum for culture and transformant is made through selection with antibiotics. The method of the invention is characterized by high regeneration rate, good repeatability and simple operation and the method lays a foundation for molecular operations such as gene expression and functional analysis of the penicillium digitatum.

Description

technical field [0001] The invention relates to a method for genetic transformation of Penicillium, in particular to a method for genetic transformation mediated by Agrobacterium tumefaciens of finger mold. Background technique [0002] There are many kinds of Penicillium fungi, some of which have important economic value in medicine and industry, such as P. chrysogenum Thom and P. Made a significant contribution; Griseofulvin can be extracted from Penicillium griseofulvum (P.griseofulvum). However, some species of Penicillium fungi are important pathogens of humans, such as Penicillium marneffei (P.marneffei), which is the culprit of deep fungal infectious diseases, and is considered to be the most common opportunistic infection among AIDS patients in Southeast Asia one. [0003] Many species of fungi in this genus are parasitic or saprophytic on fruits, vegetables, grains, meat and other foods, not only causing rot and mildew of these foods, but also endangering human he...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/11C12R1/80
Inventor 王继业李红叶张志芳
Owner ZHEJIANG UNIV
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