Method for preparing apple tree valsa ceratosperma transformant and GFP (Green Fluorescent Protein) labelled strain

A technology for rot bacteria and apple trees, which is applied in the field of apple tree rot bacteria GFP marker strain and preparation thereof, apple tree rot bacteria transformants and their preparation fields, and can solve the problem of low transformation efficiency, no rot bacteria GFP labeling method, lack of high efficiency, Problems of stabilizing the genetic transformation technology system of apple tree rot fungus

Active Publication Date: 2013-03-27
QINGDAO AGRI UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

In response to this problem, many scholars have proposed some improved technologies and new methods, the most effective of which is Agrobacterium tumefaciens-mediated transformation (ATMT), which is used in the genetic transformation of various filamentous fungi. Gao Jing (2011, master's thesis) initially established the ATMT transformation system of apple tree rot pathogen, but the transformation efficiency is extremely low, every 10 6 Only 6 transformants were obtained from each conidia
[0007] In short, there is currently a lack of an efficient and stable genetic transformation technology system for the rot pathogen of apple trees, and there is no report on the GFP labeling method of the rot pathogen, which has become a bottleneck for the research on the pathogenic process and mechanism of the rot pathogen infection.

Method used

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  • Method for preparing apple tree valsa ceratosperma transformant and GFP (Green Fluorescent Protein) labelled strain
  • Method for preparing apple tree valsa ceratosperma transformant and GFP (Green Fluorescent Protein) labelled strain
  • Method for preparing apple tree valsa ceratosperma transformant and GFP (Green Fluorescent Protein) labelled strain

Examples

Experimental program
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Effect test

example 1

[0089] Example 1: Construction of the T-DNA random insertion mutant library of the apple tree rot fungus bacterial strain LXS080901

[0090] 1. Preparation of conidia of transformed recipient apple tree rot fungus LXS080901

[0091] The preserved pathogenic rot strain LXS080901 was inoculated in PDA medium, and cultured in constant temperature and dark at 25°C for 3 days. Inoculate the activated apple tree rot pathogen into a cake (diameter 6mm) on the plates prepared by the following 6 media, and continue to culture in a 25°C incubator in the dark. When inducing sporulation in the sixth medium described below, put sterilized apple branches into the sterilized apple branches after the mycelium of the rot pathogen grows on the PDA medium, and place them under a black light (365nm) for induction. The formulations of the 6 media are as follows:

[0092] (1) PDA medium Weigh 200g of peeled potatoes, add an appropriate amount of distilled water to boil for 30 minutes, filter with...

Embodiment 2

[0156] Example 2: Observing the infection process of pathogenic bacteria using the GFP-labeled strain of putrefactive bacteria LXS080901

[0157] 1. Acquisition of the rot pathogen LXS080901GFP marker strain

[0158] The constructed expression vector pHG-C, which uses the hygromycin resistance marker gene as a selection marker and constitutively expresses egfp, was transformed into Agrobacterium, followed by the method described in Example 1 to obtain a large number of transformants. Pick the transformants, and observe under a fluorescent microscope (Leica DM2500) whether the mycelium of each transformant can produce green fluorescent protein under the excitation of a 488nm blue excitation light source. The results showed that 96.7% of the picked transformants could express strong green fluorescence, and the fluorescence observation results of some transformants can be seen in Image 6 .

[0159] 2. Detection of the genetic stability of the rot pathogen LXS080901GFP-marked s...

Embodiment 3

[0168] Embodiment 3: Feasibility analysis embodiment

[0169] Utilize the method provided by the present invention, set up the genetic transformation system of multiple filamentous fungi such as pear tree rot fungus (Valsa mali), apple anthracnose fungus (Colletotrichum gloeosporioides), cherry dry rot fungus (Phomopsis perniciosa), prove that the present invention provides The stability and range of application of the method. Taking pear tree rot fungus and cherry dry rot fungus as examples, the Agrobacterium-mediated fungal genetic transformation method provided by the present invention will be described in detail below.

[0170] 1. Genetic transformation system of Agrobacterium-mediated pear rot pathogen

[0171] The activated pear rot fungus LXS240101 was inoculated onto PDA medium, cultured in dark at a constant temperature at 25°C until conidia were produced, and the conidia were picked and diluted to 10 with Agrobacterium EHA105 containing plasmid pBIG3C. 6 cells / mL, ...

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Abstract

The invention aims to provide an apple tree valsa ceratosperma transformant and a preparation method thereof. The method comprises the following steps of: after mixing, co-culturing conidial suspensions of agrobacterium tumefaciens containing a binary vector and valsa ceratosperma, screening by using antibiotics to obtain the transformant. According to the method, the conidium of apple tree valsa ceratosperma is used as a receptor, agrobacterium tumefaciens is used as an amboceptor, the problems of difficulty in preparation of protoplast of the apple tree valsa ceratosperma and difficulty in genetic transformation of the valsa ceratosperma caused by low polyethylene glycol (PEG)-mediated transformation efficiency and the like are overcome; the transformation efficiency of the method can be up to 1/10<3> conidiums; simultaneously, the invention provides an apple tree valsa ceratosperma green fluorescent protein (GFP) labelled strain and a preparation method thereof; and the efficiency of the green fluorescent protein with strong expression in the transformant obtained by the method can be up to 96.7%.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, specifically relates to a transformant of apple tree rot pathogen and a preparation method thereof, and also provides a GFP-marked bacterial strain of apple tree rot pathogen and a preparation method thereof. Background technique [0002] Apple tree rot disease caused by Valsa ceratosperma is the most destructive disease that threatens the apple industry. Garden (Chen Ce, 1987; 2009). Since apple tree rot disease was discovered in the southern part of Liaoning Province in 1916, the disease has had several epidemics in my country, causing serious yield and economic losses (Chen Ce et al., 1980; 2009). With the adjustment of my country's apple planting structure and cultivation system, rot disease has become the main factor restricting the development of my country's apple industry. In 2008, the National Apple Industry Technology System Survey showed that the overall inciden...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12R1/645
Inventor 王彩霞李保华王海艳李桂舫董向丽张清明
Owner QINGDAO AGRI UNIV
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