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Microplitis mediator MmedCO I gene and application thereof

A ditch wasp and gene technology, applied in the field of MmedCOⅠ gene of the mid-red side ditch wasp, can solve the problems of increasing investigation time and cost, abnormal death of host larvae, affecting statistical results, etc., so as to shorten the detection time and avoid statistical errors. , the effect of improving the accuracy

Active Publication Date: 2016-08-17
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, this method requires the host larvae to be reared continuously until the parasitoid cocoons, which increases investigation time and cost
In addition, parasitic wasps need a long time to develop in the body of the host larvae, and the abnormal death of the host larvae during the development process will eventually affect the statistical results

Method used

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  • Microplitis mediator MmedCO I gene and application thereof
  • Microplitis mediator MmedCO I gene and application thereof
  • Microplitis mediator MmedCO I gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Obtaining CO Ⅰ Gene of Zhonghong Sidegou Cocoon Peak

[0033] Log in to the NCBI website to download the registered nucleic acid sequence of the CO Ⅰ gene of Hymenoptera insects, use this sequence as the "Query" sequence, and use the local BLAST function of the BioEdit software to search for the transcriptome of the Braconis spp. The homologous sequence of the CO Ⅰ gene was obtained from the database, named Mmed CO Ⅰ.

[0034] The target gene sequence was compared with the NCBI non-redundant protein database, and it was found that the target gene had a high sequence similarity with insect CO Ⅰ family members, and the amino acid sequence identity with Cotesiavestalis CO Ⅰ was 79%. Therefore, the Mmed CO Ⅰ gene was judged to be the CO Ⅰ gene.

Embodiment 2

[0035] Example 2 Specific primers for amplifying the Mmed CO Ⅰ gene

[0036] Specific primers were designed by downloading the CO Ⅰ gene homologous sequences of host and non-host insects such as cotton bollworm, armyworm, and beet armyworm, and using the clustalx2.0 software to perform homologous comparison, and search for more than 2 according to the comparison results. The region with different bases is used as the 3' end sequence of the forward and reverse primers. In order to increase the specificity of the primers, the length of the primers should be more than 20 bases, the Tm value should be higher than 55°C, and the size of the amplified product fragment should be controlled at about 100-300bp According to the above principles, a total of 3 pairs of primers were designed, and only one pair of primers was found to be specific by PCR detection:

[0037] MmedCOI-F: 5'-ATTCGTTTGGAATTAGGTATACCAGGTAG-3';

[0038] MmedCOI-R: 5'-ACAGTTCATCCAGTTCCAAACACCAAC-3'.

[0039] Only u...

Embodiment 3

[0040] Example 3 Specificity and Sensitivity Test of Specific Primers

[0041] Specific detection:

[0042] The genomic DNAs of cutworm, yellow cutworm, armyworm, cotton bollworm, beet armyworm, and red cutworm were extracted respectively, and whether there were non-specific bands were detected by PCR method, such as figure 1 shown. The results showed that only the DNA sample of Brachia schrenzo had a specific band, and the sequencing results showed that the band was the target sequence.

[0043] Sensitivity test:

[0044] Dilute the DNA samples of Braconis sclerosus to different concentrations, and detect the PCR products of DNA samples at different concentrations, such as figure 2 shown. The results showed that the target sequence could be effectively detected even if the DNA sample content of Brachia schrenzia was 0.01ng.

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Abstract

The invention relates to the field of parasitoid detection, and specifically provides a Microplitis mediator MmedCO I gene sequence (as shown in SEQ ID No.1), and specific amplification primers, which are designed based on the sequence, and provides a molecular level method for rapid detection of parasitic cotton bollworm larvae of the Microplitis mediator. The invention facilitates quick and accurate calculation of the egg laying amount of parasitic wasps or parasitism rate of host larvae. The invention can effectively detect the parasitized state of host larvae 24 hours after parasitism of the host larvae, reduce detection time, avoid statistical error due to abnormal death of host larvae and improve the accuracy of statistics.

Description

technical field [0001] The invention relates to the field of parasitoid detection, in particular to a MmedCO Ⅰ gene of Braconis sclerosus and its application. Background technique [0002] Microplitis mediator (Haliday) is a larval parasitoid with a wide range of hosts, involving more than 40 kinds of insects such as Lepidoptera Noctuidae and Geometridae, including cotton bollworm (Helicoverpa armigera), armyworm (Mythimna separata) and small cutworm (Agrotis ypsilon), which are important agricultural pests, play an important role in integrated pest management strategies. The parasitic effect of B. chinensis on host larvae is high in the field, and the survey found that the average natural parasitism rate of B. chinensis on each generation of cotton bollworm can reach 22.9%. [0003] In the study of parasitic wasp parasitic behavior, the parasitism rate of parasitoid host larvae parasitized by parasitoids is an important content of the research. Traditional statistical meth...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/11C12Q1/68
Inventor 王山宁张永军郑瑶彭勇路子云郭予元
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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