Method for constructing fluorescence labeled rhizobia

A technology of fluorescent labeling and construction method, which is applied to the construction field of fluorescently labeled rhizobia, can solve the problems of bacterial proliferation speed and conjugation transfer rate limitation, etc., and achieves the effects of cheap and easy-to-obtain raw materials, sufficient raw materials, and simple process methods.

Inactive Publication Date: 2013-11-27
GANSU AGRI UNIV
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Problems solved by technology

However, due to the artificially prepared single-layer filter membrane structure, the nutrients in the medium can only be abso

Method used

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Experimental program
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Embodiment 1

[0034] Treatment of alfalfa leaves:

[0035] Take the young leaves of alfalfa (area 1.5cm 2 -2.5cm 2 , thickness 0.03mm-0.04mm), after cleaning with distilled water, place the leaves evenly in a single layer in a 90mm culture dish, and sterilize at 121°C for 26 minutes; take out the leaves with sterile tweezers, stick them on the surface of TY medium, and cover them Petri dish so that the water will not be lost too much.

[0036] The improved splicing transfer process is as follows:

[0037] Step 1: Streak activation of donor bacteria and auxiliary bacteria on LB solid medium containing 40 μg / mL gentamicin and 20 μg / mL spectinomycin; activate recipient bacteria on TY solid medium.

[0038] Step 2: Pick the activated donor bacteria and auxiliary bacteria, transfer them to 50ml LB medium containing 40μg / mL and 20μg / mL respectively, and cultivate them to the logarithmic phase (OD 600nm =0.3-0.5). The recipient bacteria were inserted into 50mLTY liquid medium and cultured...

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Abstract

The invention discloses a method for improving conjugational transfer efficiency of fluorescent plasmid transfer of fluorescence labeling strains of rhizobia by taking sterilizing alfalfa leaves as triparental mating carriers instead of sterile filter membranes. The method can be used for efficiently constructing CFP (cyan fluorescent protein) gene labeled strains of rhizobia. The method specifically comprises the following steps of taking small and tender leaves of alfalfa, uniformly putting the leaves in a single layer in a culture dish after washing the leaves with distilled water, and sterilizing the leaves; taking out the leaves with a pair of sterile tweezers, sticking the leaves to the surface of TY culture media, and covering the culture dish; and using the leaves to replace microporous filter membranes in the triparental mating process to carry out mixed growing culture on donors, assistant bacteria and recipient bacteria. Compared with the microporous filter membranes as the mating carriers, the alfalfa leaves, as the mating carriers, can improve the conjugational transfer efficiency of the fluorescent plasmids by 8.4-11 times, and the difference is obvious. As the used raw materials are alfalfa plant leaves which are renewable and accessible, the method has the advantage that the raw materials are cheap and accessible, is simple and convenient to operate and is suitable for constructing the fluorescence labeling strains of rhizobia.

Description

technical field [0001] The invention relates to the field of crop inoculants, in particular to a method for constructing fluorescently labeled rhizobia. Background technique [0002] Rhizobia inoculants are commonly used in agricultural production, and the yield of leguminous crops has also been significantly increased due to the application of rhizobia inoculants. also needs to be resolved. The development of rhizobia labeling technology and its application in scientific research and practical production have gradually clarified the nitrogen fixation mechanism of rhizobia and the process of infection and colonization in the roots of leguminous plants. One of the effective methods of competition and interaction with other microorganisms in the body. At present, the labeling effect is the best, and the easiest and quickest method for determination is fluorescent protein labeling of rhizobia. The CFP (Cyan Fluorescent Protein) gene is a mutant extracted from the jellyfish g...

Claims

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Application Information

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IPC IPC(8): C12N15/03
Inventor 师尚礼陈力玉李剑峰张淑卿曹文侠霍平慧苗阳阳
Owner GANSU AGRI UNIV
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