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Primer pair for assaying expression of CG5210 gene of tephritidae

A technology of CG5210 and gene expression, which is applied in the field of primer pairs for detection of CG5210 gene expression in fruit flies, can solve problems such as fungal infection, fruit rot and fall off, and achieve the effects of low cost, simple operation, and controlled diffusion

Active Publication Date: 2018-12-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The harm is that adults lay eggs on the inner side of the peel, and the larvae eat the fruit, causing fungal infection and causing the fruit to rot and fall off

Method used

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  • Primer pair for assaying expression of CG5210 gene of tephritidae
  • Primer pair for assaying expression of CG5210 gene of tephritidae
  • Primer pair for assaying expression of CG5210 gene of tephritidae

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1. Preparation of primer pairs for detection of fruit fly CG5210 and Idgf4 gene expression

[0040] This embodiment provides a pair of primers for detecting the expression levels of CG5210 and Idgf4 genes in fruit flies, which are respectively denoted as CG5210-rt and Idgf4-rt. CG5210-rt is composed of two single-stranded DNAs shown in sequence 1 and sequence 2 in the sequence listing, the molar ratio of the two single-stranded DNAs in the primer pair is 1:1, and the two single-stranded DNAs are packaged independently. Idgf4-rt is composed of two single-stranded DNAs shown in sequence 3 and sequence 4 in the sequence listing, the molar ratio of the two single-stranded DNAs in the primer pair is 1:1, and the two single-stranded DNAs are packaged independently.

Embodiment 2

[0041] Example 2, using the primer pair of Example 1 to detect the expression of CG5210 and Idgf4 genes in fruit flies

[0042] Fruit flies to be tested: Guava fruit flies collected from Yunnan and Bactrocera dorsalis collected from Guangzhou, 50 first-instar larvae, 30 second-instar larvae, and third-instar early larvae (5 days after egg hatching) 30 instar larvae), 20 late third-instar larvae (7-day-old larvae after egg hatching), 6 early-stage pupae (1-day-old pupae since pupation), 6 mid-stage pupae (5-day-old pupae since pupation) 6, 6 late stage pupae (9 days old pupae since pupation), 10 immature adults after eclosion, 10 sexually mature adults 10 days old.

[0043] The total RNA of each fruit fly was extracted using the RNAsimple Total RNA Extraction Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., and then reverse-transcribed to obtain cDNA. Using the cDNA obtained by reverse transcription of Bactrocera dorsalis and Bactrocera guava as templates, the Takara...

Embodiment 3

[0054] The primers of embodiment 3, embodiment 1 are to the optimization of required template amount

[0055] Carry out 10-fold gradient dilution respectively to the cDNA that embodiment 2 obtains, obtain the cDNA dilution liquid of the dilution 10 times, 100 times, 1000 times and 10000 times of the specific development stage of each fruit fly (the content of cDNA is respectively 100 μ g / μl, 10 μ g / μl, μl, 1 μg / μl and 0.1 μg / μl), and then use this as a template to determine the optimal template amount using the reaction system and reaction procedure in Example 2, and use water to set up a negative control.

[0056] The results showed that using 100 μg / μl, i.e. 10-fold diluted cDNA, as a template, both primer pairs could obtain positive amplification curves in B. dorsalis and B. guava, and the amplification curves of internal reference genes and target genes The CT value of the target gene is within the reasonable range of 20-30; while 10 μg / μl, 1 μg / μl and 0.1 μg / μl cDNA as t...

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Abstract

The invention discloses a primer pair for assaying the expression of the CG5210 gene of tephritidae. The primer pair disclosed by the invention consists of two single-stranded DNAs shown as sequence 1and sequence 2 in a sequence table. An experiment proves that the primer pair for assaying the expression of the CG5210 gene of tephritidae can specifically, rapidly and accurately carry out quantitative assay for the CG5210 gene of bactrocera dorsalis and bactrocera correcta, and can be used for evaluating the interfering efficiency of the CG5210 gene of tephritidae. In addition, quantitative assay solves the problem on how to compare the expressions of the same gene between different tephritidae, moreover, operation is convenient and efficient, and the cost is low. The invention can also provide tools and related targets for the research of the CG5210 gene and the prevention and control of tephritidae, having important significance for the prevention and control of tephritidae, and moreover, the invention provides a potential target genet reference for the effective control of the spread of dangerous tephritidae, the protection of agricultural production and ecological safety and the promotion of the economic and trade development of China.

Description

technical field [0001] The invention relates to a pair of primers used for detecting the expression level of the fruit fly CG5210 gene in the field of biotechnology. Background technique [0002] Bactrocera dorsalis (Hendel) belongs to Diptera, Tephritidae Tephritidae, and Bactrocera genus Bactrocera. It is a polyphagous pest and can eat more than 250 kinds of fruits and vegetables in 46 families such as citrus, guava, and mango. The larvae feed on the inside of fruits and vegetables and the adults lay eggs on the fruit skin to form holes, causing fruit drop or whole fruit to rot. This not only brings serious harm to fruit and vegetable production, but also has a serious impact on import and export trade. Due to its wide host range, high fecundity and strong adaptability, many countries list it as a key quarantine object (Zhang Bin et al., 2008). [0003] Bactrocera dorsalis is native to the tropical regions of Asia and now has a wide distribution, involving tropical, subtr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/158C12Q2531/113C12Q2563/107C12Q2545/101
Inventor 李志红顾欣悦柳丽君赵岩
Owner CHINA AGRI UNIV
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