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Method for constructing molecular map of bactrocera dorsalis intestinal bacteria colony

A technology of intestinal bacteria and Bactrocera dorsalis, applied in the field of bioengineering, can solve problems such as unclear microbial community structure and achieve important application value

Inactive Publication Date: 2015-03-25
江西省农业科学院农业应用微生物研究所
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Previous studies on the intestinal flora of Bactrocera dorsalis and its attracting performance have all used traditional microbiological methods. The structure of the overall microbial community in the intestinal tract of Bactrocera dorsalis and whether the bacteria that cannot be cultured in the intestinal tract also have an attractive effect are still unclear. don't know

Method used

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  • Method for constructing molecular map of bactrocera dorsalis intestinal bacteria colony
  • Method for constructing molecular map of bactrocera dorsalis intestinal bacteria colony
  • Method for constructing molecular map of bactrocera dorsalis intestinal bacteria colony

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of the 16S rDNA library of Bacteralis dorsalis intestinal bacterial community

[0035] 1. Collection of samples

[0036] The adults used in the experiment came from three populations, namely the laboratory population (lab-reared (LR)), the laboratory sterile sugar-reared (LSSR) population and the wild population (field-collected (FC)) .

[0037] The laboratory population and the laboratory population fed with aseptic sugar water come from this laboratory, and the breeding conditions in the insectarium are: temperature 27°C±1°C, relative humidity 70%-80%, and the light cycle is natural light source. The laboratory population was fed with sucrose:yeast powder=1:3, and the sterile sugar water fed population was fed with 20% sucrose solution (sterilized by filtration). The wild population was collected from the orangery of the cadre rest center of the Bayi Road Military Region, Wuchang District, Wuhan, and brought back to the laboratory for intestinal...

Embodiment 2

[0060] Phylogenetic Analysis of Example 2 Bacterial 16S rDNA Library

[0061] For the migratory position of the PCR amplification product obtained in Example 1 on the DGGE gel, select different recombinant plasmids (by visual observation, the bands at the same migratory position are classified as the same recombinant plasmid, which is the same phylogenetic type (phylotypes) or operational taxonomic units (OTUs)).

[0062] According to the DGGE electrophoresis pattern, 55, 26 and 69 different recombinant plasmids were screened out from 178, 181 and 187 recombinant plasmids in the three populations, and their inserts were sequenced (entrusted to Beijing Aoke Biotechnology Co., Ltd.) . Submit each sequencing result to the GENBANK nucleic acid sequence database for comparison with known sequences, and construct a phylogenetic tree with MEGA4.1. The obtained sequence was compared with the 16S rDNA gene sequence in GenBank by the BLAST program, the genetic distance was calculated ...

Embodiment 3

[0067] Diversity analysis uses the diversity index formula and Analytic Rarefaction 1.3 software to calculate the diversity index and coverage of the three libraries:

[0068] The coverage (C) was used to evaluate the expression of the constructed 16S rDNA library on environmental bacterial diversity, and the formula was:

[0069] C=1-(n1 / N)×100%,

[0070] In the formula, N represents the total number of clones in the 16S rDNA library, and n1 represents the number of OTUs that appear only once in the library.

[0071] For the obtained library 16S rDNA sequences, Shannon-Weiner index and Simpson index were used to describe the diversity of the community. The formula for the Shannon-Wiener index is:

[0072] H=-∑(P i )(㏑P i )

[0073] In the formula, Pi is the ratio of the number of clones of each OTU in the library to the total number of clones in the library.

[0074] The formula for the Simpson index is:

[0075] D=1-∑(P i ) 2

[0076] The Chao-1 index was used to e...

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Abstract

The invention relates to a method for constructing a molecular map of a bactrocera dorsalis intestinal bacteria colony. The method comprises the following steps: extracting metagenomic DNA of bactrocera dorsalis intestinal bacteria as a template, amplifying V6-V8 hypervariable regions of 16S rDNA gene by using primer pairs 968GC and L1401, recovering an amplification product to obtain a DNA fragment library, connecting with a pMD19-T vector to obtain a recombinant plasmid library, amplifying the recombinant plasmid by using a primer pair comprising a forward primer RV-M and a reverse primer M13-47, amplifying a positive recombinant plasmid by 968GC and L1401 to obtain V6-V8 hypervariable regions of the positive recombinant plasmid 16S rDNA gene, and performing denaturing gel gradient electrophoresis to obtain the molecular map. The method is simple and practical and used for constructing the bacteria colony 16S rDNA library, and has important meaning on definition of the structure, inheritance and functional diversity of the bactrocera dorsalis intestinal bacteria colony.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for constructing a molecular map of intestinal bacterial communities of Bactrocera dorsalis. Background technique [0002] Bactrocera dorsalis (Hendel), also known as Bactrocera orientalis, belongs to the genus Bactrocera of the family Diptera Tephritidae. The insect is now widely distributed in many countries and regions in Asia and the Pacific Rim. With the global climate change and the increasing frequency of international trade, it has become a dangerous fruit and vegetable pest in South China and Southwest my country. Because of its strong reproductive ability and wide range of hosts, it can harm more than 250 kinds of fruits and vegetables in more than 40 families such as citrus, peach, mango, banana, eggplant, pepper, and melon. It is known as the "number one killer" of fruits and vegetables. It is an important quarantine pest, and my country on...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q2565/125C12Q2563/173
Inventor 王洪秀张宏宇靳亮魏云辉陈庆隆
Owner 江西省农业科学院农业应用微生物研究所
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