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Method for quantitatively detecting endophytic bacteria of plant tissue by non-culture method

A technology of plant tissue and endophytic bacteria, applied in the biological field, can solve the problems of unknown abundance of non-culturable bacteria, quantitative error of plant endophytic bacteria, etc.

Pending Publication Date: 2022-01-21
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These studies have quantified the endophytic bacteria in different tissues of plants, however, the quantification of plant endophytic bacteria in these studies is based on the cultivable method of isolating endophytic bacteria and then counting colonies
However, on the one hand, due to the limitations of microbial isolation and culture techniques, it is difficult to ensure that all culturable bacteria in plant tissues are isolated and cultured in a study, which leads to errors in the quantification of plant endophytes by culture methods; on the other hand, In nature, most bacteria remain unculturable, and the abundance of unculturable bacteria inside plant tissues remains unknown

Method used

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  • Method for quantitatively detecting endophytic bacteria of plant tissue by non-culture method
  • Method for quantitatively detecting endophytic bacteria of plant tissue by non-culture method
  • Method for quantitatively detecting endophytic bacteria of plant tissue by non-culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, plant callus total DNA extraction experimental scheme

[0031] 1) Take about 50 mg of callus (callus obtained by inducing dedifferentiation using rice seeds as material) and place it in a 2ml centrifuge tube, add liquid nitrogen and grind thoroughly.

[0032] 2) Add 567 μl of TE buffer solution (recipe: solvent is water, solute is 10 mM Tris, 1 mM EDTA; pH 8.0), vortex, and mix thoroughly.

[0033] 3) Add 30 μl of 10% (10 g / 100 ml) SDS and 20 μl of proteinase K (100 μg / ml, AMRESCO, Solon, USA) (equivalent to 0.76 U of proteinase K), vortex, and mix well.

[0034] 4) Add 2 μl of RNase (100 mg / ml, Tiangen Biochemical Technology Co., Beijing), mix by inversion and incubate at 37° C. for 1 hour.

[0035] 5) Add 100μl 5M NaCl, mix by inversion, then add 80μl CTAB / NaCl solution (recipe: solvent is water, solute is 0.7M NaCl, 10% (10g / 100ml) CTAB), incubate at 65℃ after inversion 10min.

[0036] 6) Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:...

Embodiment 2

[0042] Embodiment 2, making the standard curve of plant tissue absolute bacterial content determination

[0043] The callus is cultured from the surface-sterilized plant tissue in a sterile environment, therefore, the default callus is sterile. The present invention is based on the DNA of callus (the callus obtained by inducing dedifferentiation using rice seeds as materials), adding different copy number gradients of E. coli genomic DNA as a template, and then using the 322F-1 / 796R primer pair for fluorescence Quantitative PCR (qPCR), and draw a standard curve of plant tissue bacterial content and qPCR reaction Ct value according to the results.

[0044] 322F-1: 5'-ACGGHCCARACTCCTACGGAA-3' (SEQ ID No. 1);

[0045] 796R: 5'-CTACCMGGGTATCTAATCCKG-3' (SEQ ID No. 2).

[0046] Wherein, H represents A or C or T; R represents G or A; M represents A or C; K represents G or T.

[0047] Specific steps are as follows:

[0048] 1. Streak culture of E.coli DH5α strain on LB plate, 37°...

Embodiment 3

[0061] Embodiment 3, rice tissue endophytic bacteria absolute content determination

[0062] The standard curve that embodiment 2 draws can calculate the bacterial content of unit weight plant tissue by weighing the plant tissue that is used for DNA extraction, the present invention is example with model plant rice, to rice root, leaf two kinds of tissues The absolute content of endophytic bacteria was determined.

[0063] 1) Disinfection on the surface of rice tissue: first wash with 75% (volume percentage) ethanol for 5 minutes, then transfer the rice leaves to sterile water (ddH 2 O and Tween20 were added at a volume ratio of 1000:1, vortexed for 2-5min), and finally sterilized with ddH 2 O wash 4 times.

[0064] 2) The rice tissue DNA extraction method is the same as in Example 1.

[0065] 3) The qPCR reaction (BIO RAD) system is: 2×iQTM SYBR Green Mix 12.5 μl; template 1 μl (rice root or leaf tissue DNA); primers 322F-1 and 796R (see above for specific sequences) 0.5 μ...

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Abstract

The invention discloses a method for quantitatively detecting endophytic bacteria of plant tissues through a non-culture method. The method comprises the following steps: adding bacterial DNA with different copy numbers into genome DNA extracted from sterile calluses, and carrying out fluorescent quantitative PCR (Polymerase Chain Reaction) by using a bacterial 16S rRNA gene specific primer pair to obtain a standard curve of the plant tissue bacterial content and a fluorescent quantitative PCR reaction Ct value; carrying out surface sterilization on plant tissues to be detected, and extracting total DNA; and taking the extracted DNA as a template, carrying out fluorescent quantitative PCR (Polymerase Chain Reaction) by adopting the bacterial 16S rRNA gene specific primer pair, and substituting the obtained Ct value into the standard curve to obtain the content of the endophytic bacteria in the plant tissue to be detected. The method is easy and convenient to operate, time-saving, efficient, capable of covering all endophytic bacteria of plant tissues and suitable for various plants, and the bacterial content of the plant tissues to be detected can be directly obtained by substituting the qPCR reaction Ct value of a sample to be detected into the standard curve in related research.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for quantitatively detecting endophytic bacteria in plant tissues through a non-culture method. Background technique [0002] Plant endophytic bacteria are defined as cultivable and nonculturable bacteria that are planted inside plant tissues or isolated from surface sterilized plant tissues and are harmless to plants. Due to the colonization inside the tissue, the endophytic bacteria are in close contact with the plant, and the interaction with the host plant is more direct and efficient, and they are an important part of the plant microbiome. Studies have found that a variety of endophytic bacteria show beneficial effects on plants, mainly including three aspects: promoting the nutrient absorption of plant nitrogen, phosphorus, ions, etc.; regulating plant hormones such as auxin, cytokinin, and ethylene to regulate plant growth and development. Growth and development; hel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/06
CPCC12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 张莉莉方荣祥陈丽莹
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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