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Method of analyzing rice phyllospheric endophytic bacteria community structure

A technology of endophytic bacteria and endophytic bacteria, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve scientific problems that affect data analysis and analysis quality

Active Publication Date: 2017-07-21
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The determination of endophytic bacteria in the rice phyllosphere also requires 16S rDNA sequencing, but the problem is as mentioned above, the presence of mitochondrial DNA and chloroplast DNA in rice mesophyll cells makes these two types of interference account for more than 90% of the amplification products. The sequence information of the bacteria is almost covered, which seriously affects the analysis of the sequenced data and the scientific quality of the analysis

Method used

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  • Method of analyzing rice phyllospheric endophytic bacteria community structure
  • Method of analyzing rice phyllospheric endophytic bacteria community structure
  • Method of analyzing rice phyllospheric endophytic bacteria community structure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, rice and its phyllospheric endophyte total DNA extraction experimental scheme

[0049] 1) Preparation: The mortars, hammers, centrifuge tubes, tweezers, etc. used in the experiment were all sterilized by high pressure steam in advance.

[0050] 2) Removal of bacteria on the surface of rice leaf tissue: Soak the rice leaves frozen at -80°C in 75% (volume percent Hanlang) ethanol for 5 minutes, and then transfer the rice leaves to a place where the surfactant Tween20 (AMRESCO , Solon, USA) 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.

[0051] 3) After fully grinding with liquid nitrogen, take about 100mg and place it in a 2ml centrifuge tube.

[0052] 4) 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.

[0053] 5) Add 30 μl of 10% (10 g / 100 ml) SDS and 20 μl of proteinase K ...

Embodiment 2

[0062] Embodiment 2, nested PCR amplification

[0063] 1. Template: the rice leaves extracted by the method of Example 1 and the total DNA of endophytes

[0064] 2. Enzyme: KOD Plus (Toyobo, Japan)

[0065] 3. Primers (concentration 10 μM): 329F / 1417R, 799F / 1193R

[0066] 329F: 5'-ACGGHCCARACTCCTACGGGA-3' (SEQ ID NO: 1);

[0067] 1417R: 5'-GTGTACAAGRCCCGGGAACG-3' (SEQ ID NO: 2).

[0068] 799F: 5'-AACMGGATTAGATACCCKG-3' (SEQ ID NO: 3);

[0069] 1193R: 5' - ACGTCATCCCCACCCTTCC-3' (SEQ ID NO: 4).

[0070] Wherein, H represents A or C or T; R represents A or G; M represents A or C; K represents G or T. The amplified product of primer 329F / 1417R is the V3-V8 region of 16S rDNA, and the amplified product of primer 799F / 1193R is the V5-V7 region of 16S rDNA.

[0071] 4. Nested PCR

[0072] (1) The first round of PCR amplification (1 st PCR)

[0073] Reaction system: 10×KOD Buffer 5μl; 2mM dNTP 5μl; MgSO 4 2μl; KOD Plus 1μl; primers 329F and 1417R each 1.5μl; template (tota...

Embodiment 3

[0080] Embodiment 3, the sequencing identification of amplified product

[0081] 1. Monoclonal sequencing method

[0082] 1. Recovery and purification of amplification products

[0083] (1) After 1% agarose gel electrophoresis, the gel strip containing the target band was excised and placed in a 1.5ml centrifuge tube (AXYGEN).

[0084] (2) Use Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA) kit for recovery and purification, and operate according to the instructions.

[0085] (3) Finally add ddH preheated at 65°C 2 O 20 μl for elution, recover and purify the amplified product and store it at -20°C.

[0086] 2. The amplified product was connected to an empty vector to construct a complete plasmid

[0087] Target fragment: the high-fidelity PCR product of KOD Plus enzyme, that is, the amplification product of nested PCR.

[0088] Vector: pEasy Blunt Simple Vector (Quanshijin, Beijing, China)

[0089] Ligation system: vector 1 μl; target fragment 4 μl.

[009...

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Abstract

The invention discloses a method of analyzing rice phyllospheric endophytic bacteria community structure, comprising the steps of a) extracting total DNA of a plant and its phyllospheric endophytic bacteria; b) using the total DNA as a template, using a primer pair 1 (sequence 1 and sequence 2) to perform first PCR (polymerase chain reaction) amplification to obtain PCR product 1; using the PCR product 1 as a template, and using primer pair 2 (sequence 3 and sequence 4) to perform second PCR amplification to obtain PCR product 2; (c) sequencing the PCR product 2, and identifying species according to sequencing results so as to analyze the rice phyllospheric endophytic bacteria community structure. The gate to study non-culture processes of plant phyllospheric endophytic bacteria is opened, subsequent study on this field is made possible, and references are provided for the whole field of study on plant phyllospheric endophytic bacteria.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for analyzing the structure of endophytic bacterial flora in plant phyllosphere, in particular to a method for analyzing the structure of endophytic bacterial flora in rice phyllosphere by using nested PCR technology. Background technique [0002] Plant endophytes refer to microorganisms that colonize plant tissues, including endophytic fungi and endophytic bacteria. During the entire life course of plant growth and development, plant endophytes regulate the nutritional status of the host plant and participate in the host plant's response to environmental stress. (including biotic and abiotic factors). [0003] Rice is one of the most important food crops in the world. The research on rice endophytes will provide reference and guidance for rice nutrition, disease resistance and other related research. The research on plant symbiotic flora is currently mainly focused o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6869C12Q1/689C12Q2531/113
Inventor 张莉莉陈丽莹
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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