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Stably expressed reference gene group of harmonia axyridis with different factors and application of reference gene group

A technology of internal reference gene and H. axyridis, which is applied in the field of insect molecular biology research, and can solve the problems of lack of standardization research of internal reference genes for biological control of natural enemies.

Inactive Publication Date: 2018-03-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, although in RT-qPCR research, it has been proved that the systematic research on internal reference genes is very necessary, but the standardization research on internal reference genes for natural enemies is very lacking.

Method used

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  • Stably expressed reference gene group of harmonia axyridis with different factors and application of reference gene group
  • Stably expressed reference gene group of harmonia axyridis with different factors and application of reference gene group
  • Stably expressed reference gene group of harmonia axyridis with different factors and application of reference gene group

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Experimental program
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Effect test

Embodiment 1

[0046] The sequence cloning of embodiment 1 internal reference gene ACTB, GAPDH, ATP6V1A

[0047] 1. Design the degenerate primers of three internal reference genes ACTB, GAPDH, and ATP6V1A, as shown in Table 1.

[0048] Table 1: Merge primers for 3 internal reference genes ACTB, GAPDH, ATP6V1A

[0049]

[0050] 2. Gene sequence cloning

[0051] PCR amplification was carried out using the heterochromia DNA as a template, the PCR product was purified, the target fragment was recovered from agar gel, the purified product was connected to the vector pCR4TOPO (Invitrogen, Carlsbad, CA), and then transformed into Escherichia coli competent cell TOP10 , 37 ℃ plate cultured overnight, the obtained positive clones were sequenced, and the sequence obtained by sequencing was compared with the NCBI database for Blast analysis, and then the nucleotide sequence obtained was determined to be the internal reference gene sequence of the heterochromia.

Embodiment 2

[0052] Example 2 Screening of internal reference genes stably expressed in the heterochromia under different conditions

[0053] 1. The Heterochromia in this study is a laboratory-raised population with uniform feeding conditions, that is: feeding in an artificial climate chamber, feeding conditions are 23°C, 50% RH, and a photoperiod of 14L:10D. Rearing with pea aphids.

[0054] Quantitative PCR (RT-qPCR) in this study was performed on MyiQ single Color Real-Time PCR Detection System (BioRad) according to the instrument operating instructions.

[0055] 2. Processing and collection of Heterochromia under different factors

[0056] (1) Collection of Heterochromia under different temperature treatments and different photoperiod conditions

[0057] Five 3rd instar larvae were placed at 10°C, 22°C and 30°C for 3 hours, respectively.

[0058] Five third-instar larvae were placed under different photoperiods of 16L:8D, 12L:12D and 8L:16D for 2 days.

[0059] Each treatment has 3...

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Abstract

The invention discloses a stably expressed reference gene group of harmonia axyridis with different factors and application of the reference gene group. Specifically, a scheme of a stably expressed reference gene group of harmonia axyridis with different factors is that a stably expressed reference gene group under different temperature conditions comprises 18S, 28S and GAPDH (Reduced Glyceraldehyde-Phosphate Dehydrogenase), comprises 18S, 28S and HSP90 under different light cycle conditions, comprises 18S, 28S and HSP70 at different age stages, and comprises 18S, 28S and RP49 at different tissue combinations. By adopting the reference gene group, a most stable reference gene group of harmonia axyridis under four factors is confirmed for a first time, reference gene selection bases are made for study on gene expression levels of different tissues of different ages of harmonia axyridis under different temperature conditions and different light cycle conditions, and a solid base is madefor studying gene functions of the harmonia axyridis, RNAi (Ribonucleic Acid Interfere) transgenic crop environment risk estimation, and the like.

Description

technical field [0001] The invention belongs to the technical field of insect molecular biology research. More specifically, it relates to the combination of internal reference genes stably expressed by Heterochromia under different factors and their applications; including the internal reference genes stably expressed by Heterochromia at different ages, in different tissues, at different temperatures and different photoperiods, and their applications. Screening methods and applications. Background technique [0002] Harmonia axyridis is widely distributed in many countries in Asia, and then introduced or spread to Europe and America. It is widely distributed in various agricultural ecosystems around the world and is a very common predatory natural enemy insect. It is currently an important Biological defense against natural enemies is widely used in agricultural production all over the world. Its adults and larvae can prey on a variety of aphids, scale insects, psyllids, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851
CPCC12Q1/6851C12Q1/6888C12Q2600/158C12Q2600/166C12Q2531/113
Inventor 潘慧鹏杨春晓邱宝利吴建辉桑文王兴民金丰良
Owner SOUTH CHINA AGRI UNIV
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