Plant endophyte 16S rRNA gene amplification method and application

A technology of plant endophytes and plant endophytes, which is applied in the field of gene amplification to achieve the effects of reducing pollution, large amount of data and high diversity of results

Active Publication Date: 2017-01-04
成都罗宁生物科技有限公司
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Problems solved by technology

Although after years of research, the research on plant endophytic bacteria is still in its infancy. The investigation of the distribution and abundance of plant endophytic bacteria in plants still relies on traditional methods such as separation and purification using low...

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  • Plant endophyte 16S rRNA gene amplification method and application
  • Plant endophyte 16S rRNA gene amplification method and application
  • Plant endophyte 16S rRNA gene amplification method and application

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Embodiment

[0070] The total DNA of linden leaves was purified by the CTAB method, the material was pretreated according to the aforementioned method, and then the endophyte fragments of the plant were amplified according to the method of the present invention, and at the same time, without MTR (F / R) and CHP (F / R) ) The primer sample is used as a control. After the amplification is completed, the results are separated by 1% agarose gel electrophoresis. The results are as follows figure 1 Shown. The amplified bands in the control sample are mitochondrial and chloroplast bands. After using the present invention, the mitochondrial and chloroplast bands are almost invisible, and only endophytic bacterial bands are clearly visible.

[0071] After purification and recovery of the endophytic bacterial bands in plants, the aforementioned method was used to carry out secondary amplification of the 16S hypervariable region, followed by library construction and sequencing. The sequencing results are as...

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Abstract

The invention discloses a plant endophyte 16S rRNA gene amplification method and application. The amplification method includes the following steps of plant pretreating; plant-sample total DNA extracting; sample 16S rRNA gene amplifying, wherein sample 16S rRNA gene amplifying comprises 16S rRNA gene total-length emulsion PCR amplifying and 16S rRNA gene hypervariable-region/conserved-region amplifying sub-amplifying; high-throughput sequencing and biological information analyzing based on a Illumina platform, wherein high-throughput sequencing and biological information analyzing based on the Illumina platform comprises plant endophyte 16S rRNA gene amplicon purifying and recycling, amplicon sequencing library establishing, Illumina HiSeq sequencing and sequencing data bioinformatics analysis. The gene amplification method is used for high-throughput-sequencing plant disease detection and phytophagous animal enteric microorganism detection. Sequencing analysis of plant endophytic bacteria is carried out with the high-throughput sequencing technology, the data size is larger, the detection result is more complete, pollution of plant hosts is reduced to the maximum degree, the result multiformity is higher, and the cost is low.

Description

Technical field [0001] The present invention relates to the technical field of gene amplification, in particular to a method and application of 16S rRNA gene amplification of plant endophytes. Background technique [0002] Plant endophytes are an important part of the plant micro-ecosystem, and they have formed an interdependent relationship with plants in the process of long-term co-evolution. Plant endophytes can produce active substances as bio-textile resources, carriers of exogenous genes and sources of new drugs, and have great application prospects in the fields of agriculture, medicine and health. Although after years of research, the current research on plant endophytic bacteria is still in its infancy. The investigation of the distribution and abundance of plant endophytic bacteria in plants still relies on traditional methods such as separation and purification using DGGE with lower flux. Even if high-throughput sequencing is used for related research, because of the ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
CPCC12Q1/6806C12Q1/6869C12Q2531/113C12Q2535/122
Inventor 张时恒孙梓健安家兴刘驰涂波
Owner 成都罗宁生物科技有限公司
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