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Method for analyzing plant endophytic bacteria flora by primers targeting V5V6 region

A technology of plant endophytic bacteria, V5-V6, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems such as interfering flora analysis, mitochondrial sequence contamination, pollution, etc.

Active Publication Date: 2020-12-25
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
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Problems solved by technology

[0004] The plant rhizosphere flora is the most studied by using 16S amplicon sequencing to study the plant bacterial flora. For the analysis of the plant endophytic bacterial flora, the chloroplast 16S rDNA and mitochondrial 18S rDNA of plants have a high correlation with the bacterial 16S rDNA. The homology of 16S amplicon leads to the simultaneous amplification of chloroplast 16S rDNA and mitochondrial 18S rDNA by general 16S amplicon primers, and the number of these two organelles is usually far more than that of endophytic bacteria in plants. Therefore, the amplified products often Accompanied by a very high proportion of host DNA contamination, which seriously interferes with subsequent flora analysis
In the published research so far, 799F is the only primer that can avoid plant chloroplast contamination, but there is still no primer that can avoid plant mitochondrial contamination. When 799F is used in combination with other 16S amplicon primers, the amplified product is accompanied by a high proportion of mitochondrial sequence contamination

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  • Method for analyzing plant endophytic bacteria flora by primers targeting V5V6 region
  • Method for analyzing plant endophytic bacteria flora by primers targeting V5V6 region
  • Method for analyzing plant endophytic bacteria flora by primers targeting V5V6 region

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1, analyze the primer design and synthesis of plant endophytic bacterial flora

[0039] In existing studies, 799F is the only primer capable of avoiding plant chloroplast contamination, and the 799F is a single-stranded DNA molecule whose nucleotide sequence is shown in sequence 1 in the sequence listing.

[0040] In order to avoid host plant mitochondrial 18S rDNA in the present invention, primer B is designed according to the V5-V6 region of bacterial 16S rDNA as follows:

[0041] The primer B corresponds to the plant mitochondrial 18S rDNA, and the 3' end of the primer B has 3 consecutive different nucleotides from the plant mitochondrial 18S rDNA, and the full length of the primer B corresponds to the plant mitochondrial 18S rDNA There are 4 or more different nucleotides in the rDNA, except for the different nucleotides in the primer B, other nucleotides are identical or complementary to the corresponding nucleotides of the plant mitochondrial 18S rDNA. ...

Embodiment 2

[0046] Embodiment 2, the establishment of the amplification method of analysis plant endophytic bacterial flora

[0047] Using the total DNA of rice leaves as a template, primers 799F and 1107R (concentration 10 μM) described in Example 1 as primers, Platinum Taq DNA polymerase with hot start activity and no 3'→5' exonuclease correction activity (Invitrogen, USA) is an enzyme, and PCR amplification is performed to obtain a PCR amplification product.

[0048] The reaction system of above-mentioned PCR amplification is as follows:

[0049]

[0050] The flow process of the above-mentioned PCR amplification is as follows:

[0051]

[0052] The PCR amplification products were electrophoresed on 1% agarose gel, and the results were as follows: figure 1 As shown, the amplified product with a length of about 350bp was obtained, which was the V5-V6 region of the 16S rDNA of the bacterial flora.

Embodiment 3

[0053] Example 3, High-throughput sequencing method for analyzing plant endophytic bacterial flora

[0054] 1. PCR amplification:

[0055] Using the total DNA from leaves of four plants: citrus (Citrus sinensis, Cr), tomato (Lycopersicon esculentum, Le), Arabidopsis thaliana (At), rice (Oryza sativa, Os) as templates, each plant Set up 3 samples, and add 6 base lengths to the 5' ends of the primers 799F and 1107R synthesized in Example 1 to distinguish the Barcode of different samples. The primer Barcode combination of each sample is different for subsequent high-pass The sample identification of quantity sequencing; Utilize the amplification method of embodiment 2 to carry out PCR amplification, each sample obtains the amplified product of 150 μ l, amplified product is electrophoresed with 1% agarose gel, and the result is as follows figure 2 shown.

[0056] 2. Illumina sequencing results

[0057] Using the Illumina Hiseq 2500 sequencing platform of Guangdong Meige Gene T...

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Abstract

The invention discloses a method for analyzing a plant endophytic bacteria flora by primers targeting the V5V6 region. The invention provides a primer pair for analyzing a plant endophytic bacteria flora, the primer pair is composed of a primer A and a primer B, and a target sequence is the full length or part of the V5-V6 region of bacterial 16S rDNA, wherein the primer A is a single-stranded DNAmolecule with a nucleotide sequence shown as a sequence 1 in a sequence table, the primer B corresponds to plant mitochondrion 18S rDNA, three continuous differential nucleotides exist between the 3'end of the primer B and the plant mitochondrion 18S rDNA, and the number of differential nucleotides between the full length of the primer B and the plant mitochondrion 18S rDNA is larger than or equal to 4. According to the method, a pure plant endophytic bacteria flora 16S amplicon is obtained by utilizing the primer pair, and DNA polymerase which has hot start activity and does not have 3'->5'exonuclease correction activity, and a sequencing experiment platform for amplifying the endophytic bacteria flora in a pollution-free manner is established in combination with a second-generation sequencing platform.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a reagent and method for analyzing plant endophytic bacterial flora, in particular to a method for analyzing plant endophytic bacterial flora with V5V6 region primers. Background technique [0002] The surface and body of plants are colonized by a wide variety of tiny organisms, including bacteria, fungi, archaea, nematodes, protozoa, etc., which are collectively referred to as plant microbial communities. The plant microbial community is symbiotic with the host plant and affects the host plant in three aspects: vegetative growth, development and stress resistance. Bacteria are the most numerous in the plant microbial community, and they are defined as plant surface flora and plant endophytic flora according to the bacteria colonizing on the surface or inside of plant tissues. [0003] There are two types of research methods on plant microbial communities: culture method and non-cultu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6806C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6806C12Q2531/113
Inventor 张莉莉陈丽莹张玉满方荣祥
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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