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Retitermite molecular identification method based on mitochondrial CYTB gene

A mitochondrial and termite technology, applied in the fields of molecular biology and insect classification, can solve the problems of prolonged sequence cloning analysis time, increased identification cost, complicated operation, etc., and achieve the effect of shortening identification time, short time-consuming and accurate identification.

Pending Publication Date: 2022-04-12
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the continuous development of molecular biology and the deepening of corresponding research, technicians have found in the process of practical application that it is very difficult to identify different species of termites of the same genus simply by using the COI gene as a molecular marker gene. Other molecular marker genes (such as COII gene and 16SrRNA, etc.)
Therefore, in the process of classification and identification, more than one mitochondrial gene needs to be cloned, the operation is complicated, and more primers need to be designed and synthesized for PCR reaction, the cost of identification is increased, and the sequence cloning analysis time is extended, which takes more than two days

Method used

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  • Retitermite molecular identification method based on mitochondrial CYTB gene
  • Retitermite molecular identification method based on mitochondrial CYTB gene
  • Retitermite molecular identification method based on mitochondrial CYTB gene

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Construction of a DNA barcode library for the molecular taxonomic identification of Repellent termites

[0034] 1.1 Comparison of the evolution rate of the mitochondrial-encoded protein genes of the termites

[0035] Download the existing mitochondrial genome sequences of 24 kinds of Termites termites from NCBI (see Table 4), and extract 13 protein-coding genes of different termites, namely ND1, ND2, ND3, ND4, ND4L, ND5, ND6, CYTB, COX1, COX2, COX3, ATP, and ATP8. Then use Clustal X 1.81 for homologous sequence alignment analysis, output the file in .meg format after alignment, and import it into DnaSP 6 software to calculate the non-synonymous substitution rate Ka, synonymous substitution rate Ks and Ka / Ks of each protein-coding gene The value of , in order to judge the evolution rate of the protein-encoding gene of the termite.

[0036] 1.2 Statistics on the number of base variations (S) of the mitochondrial gene CYTB of the termites

[0037] The CYTB se...

Embodiment 2

[0042] Embodiment 2: the method step of classifying with the termite CYTB gene as molecular marker

[0043] 2.1 Collection of Termite samples as an example

[0044] The termite samples to be identified used in this example were collected by the inventor's team members. The collected samples included worker ants and soldier ants of Termites reticulata, which were stored in 75% and 100% alcohol solutions at 4°C and -20°C respectively. Stored in the refrigerator, soldier ants were used for morphological taxonomic identification, and worker ants were used for molecular identification. The specific collection information of the termite samples is shown in Table 1.

[0045] 2.2 Extraction of Genomic DNA from Termites termites

[0046] Use DNA extraction kit (Nanjing Nuoweizan) to extract sample DNA, the specific steps are as follows:

[0047] 1. Take 4-6 termite worker ants samples stored in a -20°C refrigerator, put them in a mortar that has been sterilized in advance, add a small...

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Abstract

The invention relates to molecular biology and insect classification technologies, and aims to provide a reticulitermes molecular identification method based on a mitochondrial CYTB gene. The method comprises the following steps: constructing a DNA (Deoxyribonucleic Acid) bar code library for classifying and identifying reticulitermes; extracting and amplifying a CYTB gene sequence of the to-be-identified reticulitermes; and determining the type of the reticulitermes according to the comparison of the CYTB gene sequence. The specific primer is used for PCR clone sequencing of the reticulitermes mitochondrial CYTB gene, the sequencing result is used as a DNA bar code for reticulitermes molecule classification and identification, and the defects in the process of using morphological identification and using molecular markers such as COI, COII and 16SrRNA to identify the reticulitermes are overcome. Compared with the prior art, the identification method disclosed by the invention is low in identification cost, short in time consumption and more accurate in identification of the subspecies of the reticulitermes. Rapid and accurate identification of the reticulitermes is realized, and the identification time can be shortened.

Description

technical field [0001] The invention belongs to molecular biology and insect classification technology, in particular to a molecular identification method of the termite termite based on the mitochondrial CYTB gene. Background technique [0002] Termites are one of the worldwide pests, mainly distributed in tropical and subtropical regions. There are 3,108 species of termites recorded, of which 371 are harmful species, which have caused serious damage and economic losses in housing construction, agriculture, forestry, water conservancy, transportation, etc., and the world needs to spend billions of dollars every year For the prevention of termites, control management and repair and maintenance work. There are 476 species of termites in 4 families, 44 genera and 476 species of termites in China, accounting for about 1 / 10 of the termite population in the world. The discovery and distribution of termites cause an annual economic loss of about 2 billion yuan. The control of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12Q1/6806G16B20/00
Inventor 张大羽谷启娟陈桐张媚
Owner ZHEJIANG FORESTRY UNIVERSITY