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A method for analyzing plant endophytic bacterial flora

A technology of endogenous bacteria and flora in plants, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve problems such as interference diversity analysis, host DNA contamination, etc.

Active Publication Date: 2020-07-14
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For the analysis of plant endophytic flora, due to the high homology between plant chloroplast 16S rDNA and mitochondrial 18S rDNA and bacterial 16S rDNA, a very high proportion of host DNA contamination will appear in the 16S amplicon library, seriously interfere with subsequent diversity analysis

Method used

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  • A method for analyzing plant endophytic bacterial flora
  • A method for analyzing plant endophytic bacterial flora
  • A method for analyzing plant endophytic bacterial flora

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1, analyze the primer design and synthesis of plant endophytic bacterial flora

[0046] The present invention designs to avoid host plant chloroplast 16S rDNA and mitochondrial 18S rDNA, and designs primer pairs according to the V3-V4 region of bacterial 16S rDNA as follows:

[0047] The last base at the 3' end of one primer is different from the plant mitochondrial 18S rDNA, and the difference between the full length of the primer and the plant mitochondrial 18S rDNA is greater than or equal to 3; the last base at the 3' end of the other primer is different from the plant chloroplast 16S rDNA difference, and the difference between the full length of the primer and the plant mitochondrial 18SrDNA is greater than or equal to 3.

[0048] According to the above design principles, the following primer pairs were designed and synthesized:

[0049] 322F-1: 5'ACGGHCCARACTCCTACGGAA3' (SEQ ID NO: 1)

[0050] 796R: 5'CTACCMGGGTATCTAATCCKG3' (SEQ ID NO: 2);

Embodiment 2

[0051] Embodiment 2, establishment of the method for analyzing plant endophytic bacterial flora

[0052] 1. PCR amplification

[0053] Using the total DNA of healthy rice leaves collected on September 12, 2016 in the rice experimental field of Fujian Agriculture and Forestry University as a template, using the primers 322F-1 and 796R of Example 1, and having hot start activity and no 3'→5' exonuclease Correction activity of DNA polymerase Platinum Taq DNA polymerase (Invitrogen, USA) was used for PCR amplification.

[0054] The reaction system of above-mentioned PCR amplification is as follows:

[0055]

[0056] In the above reaction system, the final concentration of each primer is 0.2 μM; the final concentration of Taq enzyme is 2U / rxn; MgCl 2 The final concentration was 1.5mM.

[0057] Above-mentioned PCR reaction procedure is as follows:

[0058]

[0059] The result is as figure 1 As shown, Marker: DNA Marker II (TIANGEN), 1, 2, 3: three replicate samples, 4: empt...

Embodiment 3

[0071] Example 3, High-throughput sequencing method for analyzing plant endophytic bacterial flora

[0072] 1. PCR amplification

[0073] 1. Amplification primers

[0074] Six random bases were added to the 5' ends of the primers 322F-1 and 796R designed and synthesized in Example 1 to distinguish the Barcodes of different samples. Each sample has a different primer Barcode combination, which is used for sample identification of subsequent high-throughput sequencing. The Barcode sequence information is provided by Beijing Novogene Technology Co., Ltd., and the amplification products are also provided by Beijing Novogene Technology Co., Ltd. Subsequent library construction and on-machine sequencing were carried out; 4 amplification replicates were set for PCR of each sample, and a total of 200 μl of the final amplification product was obtained, with a total of 4 samples.

[0075] 2. Amplification

[0076] Total DNA was extracted from rice leaf samples collected in Kaifeng, H...

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Abstract

The invention discloses a method for analyzing plant endophyte colonies. In the method, a primer pair used for analyzing the plant endophyte colonies is provided, the target sequence of the primer pair is the overall length or part of the region from V3 to V4 of bacteria 16SrDNA; there are differences between the last basic group at the 3' end of one primer in the primer pair and the plant mitochondria 18S rDNA, and the number of the difference sites between the overall length of the primer and the plant mitochondria 18S rDNA is equal to or larger than 3; there are differences between the lastbasic group at the 3' end of the other primer and the plant chloroplast 16S rDNA, and the number of the difference sites between the overall length of the primer and the plant mitochondria 18S rDNA is equal to or larger than 3. The two primers 322F-1 and 796R are designed, the DNA polymerase with hot-start activity but without 3' to 5' exonuclease proofreading activity is selected to achieve theaim that the pollution of the host plant chloroplast 16S rDNA and the pollution of the host plant mitochondria 18S rDNA are avoided at the same time, and therefore, a pure bacteria colony 16S ampliconlibrary is obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for analyzing plant endophytic bacterial flora, in particular to a method for analyzing the structure of plant endophytic bacterial flora by using a primer pair 322F-1 / 796R. Background technique [0002] Under natural conditions, a large number of complex and diverse micro organisms are colonized inside and outside many organs of plants, including bacteria, fungi, archaea, protozoa, etc., which affect the growth and health of plants. Among these tiny organisms, bacteria are absolutely dominant in number. According to the colonization of these bacteria on the surface or inside of plant tissues, they are divided into two types, called plant surface flora and plant endophytic flora. [0003] There are two types of research methods for plant endophytes: culture method and non-culture method. Next-generation sequencing with the help of 16S rDNA amplicon sequencing is a classic rese...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6869C12Q1/04C12N15/11
Inventor 张莉莉陈丽莹
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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