Method for observing infection process of citrus canker pathogens in real time

A technology of citrus canker bacteria and canker bacteria, which is applied in the field of real-time observation of the infection process of citrus canker fungus, can solve the time-consuming and labor-intensive problems of scanning electron microscopy and transmission electron microscopy, and achieve the effect of easy operation

Inactive Publication Date: 2012-07-11
HUNAN AGRICULTURAL UNIV
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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the time-consuming and labor-intensive deficiencies of the prior art using scanning electron mic

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  • Method for observing infection process of citrus canker pathogens in real time
  • Method for observing infection process of citrus canker pathogens in real time

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Embodiment

[0016] This embodiment includes the following steps:

[0017] (1) Using the three-parent hybridization method, the gene contained in the broad host vector pMP2463 contained in the donor bacteria Escherichia coli DH5α egfp The gene was introduced into wild-type X. citri to obtain EGFP-labeled X. citri; the EGFP-labeled X. citri emitted green fluorescence under the excitation of ultraviolet light, and the green fluorescent signal was detected by fluorescence microscopy. The growth of X. citri marked by EGFP There is no significant difference in kinetics and pathogenicity compared with wild type;

[0018] The donor bacterium Escherichia coli DH5α (containing egfp Plasmid pMP2463, which has gentamicin resistance) was provided by the Key Laboratory of Microbiology, Huazhong Agricultural University; auxiliary bacteria Escherichia coli DH5α (containing mob The plasmid pRK2013 at the locus, which has kanamycin resistance) was provided by Professor Vittoria Catara, University of Cat...

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Abstract

A method for observing the infection process of citrus canker pathogens in real time comprises the following steps: (1) guiding an enhanced green fluorescent protein gene (egfp) on an extensive host vector pMP2463 into a wild-type citrus canker pathogen to obtain an enhanced green fluorescent protein (EGFP); (2) inoculating the obtained EGFP-marked citrus canker pathogens onto the surface of a citrus leaf in a spraying way, and inoculating the obtained EGFP-marked citrus canker pathogens into mesophyll clearances of the citrus leaf through injection; and (3) sampling by tearing leaf epidermisor by a frozen section method, and observing the number and the distribution of the citrus canker pathogens in mesophyll clearances and on the surface of the citrus leaf under a laser confocal microscope. Through the adoption of the method, the attachment, breeding and spatial distribution of the EGFP-marked citrus canker pathogens in the process of infecting citrus can be observed in real time so as to obtain interactive spatial and temporal relations of citrus canker pathogens and the citrus; and the method is easy and quick to operate.

Description

technical field [0001] The invention relates to a method for observing the infection process of canker sore citrus, in particular to a method for utilizing enhanced green fluorescent protein gene egfp The invention provides a method for marking the canker citrus fungus to realize the real-time observation of the infection process of the canker canker citrus fungus. Background technique [0002] The traditional research methods for the invasion pathway of X. citri are mainly two observation methods, scanning electron microscopy and transmission electron microscopy. However, these two methods are time-consuming, laborious, and cannot be tracked in real time. [0003] Using the green fluorescent protein gene to mark pathogenic bacteria can quickly, in situ, real-time, and live observation of pathogenic bacteria, which is conducive to observing the invasion process of pathogenic bacteria and understanding the interaction between pathogenic bacteria and hosts. [0004] At presen...

Claims

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Application Information

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IPC IPC(8): C12Q1/06C12Q1/04C12R1/64
Inventor 邓子牛刘利平李大志龙桂友李建挥
Owner HUNAN AGRICULTURAL UNIV
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