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Method for analyzing plant endophyte colonies

A technology of plant endophytic bacteria and flora, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as interference diversity analysis, host DNA contamination, etc.

Active Publication Date: 2019-05-14
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • 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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  • Method for analyzing plant endophyte colonies
  • Method for analyzing plant endophyte colonies
  • Method for analyzing plant endophyte colonies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Primer design and synthesis for analyzing 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 that of plant mitochondrial 18S rDNA, and the difference between the full length of the primer and plant mitochondrial 18S rDNA is greater than or equal to 3; difference, and the difference between the full length of the primer and the plant mitochondrial 18SrDNA is greater than or equal to 3 sites.

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

[0049] 322F-1: 5'ACGGHCCARACTCCTACGGAA3' (sequence 1)

[0050] 796R: 5' CTACCMGGGTATCTAATCCKG3' (sequence 2);

Embodiment 2

[0051] Example 2. Establishment of a method for analyzing plant endophytic bacterial flora

[0052] 1. PCR amplification

[0053] Taking the total DNA of healthy rice leaves collected in the rice experimental field of Fujian Agriculture and Forestry University on September 12, 2016 as the template, using the primers 322F-1 and 796R of Example 1, and having hot-start activity and no 3'→5' exonuclease The active DNA polymerase Platinum Taq DNA polymerase (Invitrogen, USA) was calibrated 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 is 1.5 mM.

[0057] The above 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: empty control (no ...

Embodiment 3

[0071] Embodiment 3. The method of high-throughput sequencing to analyze plant endophytic bacterial flora

[0072] 1. PCR amplification

[0073] 1. Amplification primers

[0074] 6 random bases were added to the 5' ends of primers 322F-1 and 796R designed and synthesized in Example 1 to distinguish the Barcodes of different samples. The primer barcode combination of each sample is different, which is used for the sample identification of subsequent high-throughput sequencing. The barcode sequence information is provided by Beijing Nuohezhiyuan Technology Co., Ltd., and the amplification product is also provided by Beijing Nuohezhiyuan Technology Co., Ltd. Follow-up library construction and on-machine sequencing were performed; PCR for each sample was set to 4 amplification repeats, and a total of 200 μl of final amplification product was obtained, with a total of 4 samples.

[0075] 2. Amplification

[0076] The total DNA was extracted from the leaf samples of Huangjinqing ...

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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 primer pair 322F-1 / 796R. Background technique [0002] Under natural conditions, many organs of plants are colonized with a large number of complex and diverse micro-organisms, including bacteria, fungi, archaea, protozoa, etc., which affect the growth, development and health of plants. Among these microscopic 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 categories, namely plant surface flora and plant endophyte flora. [0003] The research methods of plant endophytes include culture method and non-culture method. Next-generation sequencing by means of 16S rDNA amplicon sequencing is a classic research metho...

Claims

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Application Information

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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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