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Method for detection of hsp60 gene expression in A. zhoui by fluorescent rt-pcr technique

A RT-PCR technology, which is used in the field of detection of Hsp60 gene expression in Choweri by fluorescent RT-PCR technology, can solve problems such as influence, avoid subjectivity, avoid pollution, and improve sensitivity Effect

Inactive Publication Date: 2018-07-06
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the wild environment, C. cunea The vitality will be affected by multiple factors, among which temperature, as a main ecological factor, will affect C. cunea vitality

Method used

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  • Method for detection of hsp60 gene expression in A. zhoui by fluorescent rt-pcr technique
  • Method for detection of hsp60 gene expression in A. zhoui by fluorescent rt-pcr technique
  • Method for detection of hsp60 gene expression in A. zhoui by fluorescent rt-pcr technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035]Obtaining the Hsp60 Gene Sequence of Leptospermus zhouii

[0036] The experimental materials were obtained from the indoor subculture of the white moth A. zhouzii (cultivation conditions: in an artificial climate box (PQX-350H), temperature 25°C, relative humidity 60%-80%, no light and dark). The antennae of the female A. chowii within 24 h after eclosion were cut out under a dissecting microscope, and immediately soaked in RNAlater (Ambion, AM7020); the antennae of every 200 female A. chowii were stored in a 1.5 A total of 8 tubes of samples were collected in mL centrifuge tubes and stored at -20°C. After the samples were prepared, they were sent to Huada Gene Technology Service Co., Ltd. (http: / / www.genomics.cn / index) for transcriptome sequencing.

[0037] The high-throughput sequencing platform Illumina HiSeq™ 2000 was used to perform transcriptome sequencing on the antennae samples of Bacteria chowii. Trinity software was used to cluster each read sequence fragment a...

Embodiment 2

[0039] Extraction of total RNA

[0040] Select about 30 white moths, Chow's gnats, for different temperature treatments. The high temperature treatment (1h) is 28°C, 32°C, 36°C, 40°C and 42°C; ℃, -3℃, -7℃. The processed product was used immediately or stored frozen at -20°C.

[0041] (1) Take about 30 of the white moth Chow's gnats into a 1.5mL sterile centrifuge tube, pour liquid nitrogen into it and grind it quickly with a grinding rod. Add 600 μL Trizon solution to the centrifuge tube and let it stand for 5 minutes.

[0042] (2) Add 200 μL of chloroform into the centrifuge tube, shake vigorously for 15 seconds, and let stand for 3 minutes.

[0043] (3) Centrifuge at 12,000 rpm for 15 min at 4°C, transfer the upper colorless aqueous phase into a new sterile centrifuge tube, add 400 μL of isopropanol, place at -20°C for 20 min, and centrifuge at 12,000 rpm for 10 min.

[0044] (4) Carefully pour off the liquid in the tube, save the precipitate, and add 600 μL of 75% ethan...

Embodiment 3

[0048] cDNA synthesis

[0049] The total RNA of B. chowii described in Example 2 was used as a template. Take a sterilized 0.2mL centrifuge tube and add the following reaction system (20μL system): Total RNA 5μL, or Random Primer (N9) (0.1μg / μL) 1μL, 2×TSReaction Mix 10μL, TransScript®RT / RI Enzyme Mix 1μL , RNase-free Water 3μL, mix well.

[0050] The reaction conditions are 25°C water bath for 10 minutes, 42°C water bath for 30 minutes, and 85°C for 5 minutes to inactivate the TransScript®RT retained in the centrifuge tube. The product was used immediately or stored frozen at -20°C.

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Abstract

The invention discloses a method for detecting the relative expression quantity of Chouioia cunea Yang Hsp60 by adopting the fluorescent RT-PCR technique. According to the invention, the establishment of a real-time fluorescent RT-PCR detection method of Chouioia cunea Yang Hsp60 lays the foundation for research on the expression regulation and control mechanism of Chouioia cunea Yang Hsp60 and ecological prevention and control of Chouioia cunea Yang; the influence of temperature variation on the expression quantity of Chouioia cunea Yang in vivo Hsp60 gene is further studied, so that the research result can provide a theoretical basis for scientific utilization of natural enemy insects Chouioia cunea Yang, and further has important significance on the research that under what outdoor temperature can Chouioia cunea Yang be let to fly to achieve prevention and control of hyphantria cunea. The method further provides a technical platform for relatively quantitative analysis of Hsp60 gene at the mRNA level.

Description

[0001] The invention is funded by: National Natural Science Foundation of China (31201730); Tianjin Higher Education Science and Technology Development Project (20110602); Tianjin Normal University Doctoral Fund (52XB1003) and Tianjin Key Laboratory of Animal and Plant Resistance Open Funding. technical field [0002] The invention relates to the field of biotechnology, and relates to specific primers for detecting the expression of the Hsp60 gene of C. chowii, more specifically a method for detecting the expression of the Hsp60 gene of C. chowii using fluorescent RT-PCR technology. It is mainly used for the research on the regulation mechanism of Hsp60 gene expression and its ecological control in C. chowii. The research results can provide a theoretical basis for the scientific use of natural enemy insect C. chowii for biological control. Background technique [0003] Heat shock protein (Hsp) is a kind of highly conserved protein that exists widely in organisms and is extre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/158C12Q2600/166C12Q2561/113C12Q2545/113
Inventor 李敏张新玥王凤竹李婷婷朱耿平刘强
Owner TIANJIN NORMAL UNIVERSITY
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