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Anomala corpulenta internal reference gene and application

A technology of A. aeruginosa beetle and internal reference gene, which is applied in the direction of DNA/RNA fragments, measurement/testing of microorganisms, recombinant DNA technology, etc., to achieve the effect of improving reliability

Active Publication Date: 2018-06-29
青岛罗素生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no research report on the screening of internal reference genes of A. aeruginosa based on qRT-PCR technology and analysis of their expression stability at home and abroad.

Method used

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  • Anomala corpulenta internal reference gene and application
  • Anomala corpulenta internal reference gene and application
  • Anomala corpulenta internal reference gene and application

Examples

Experimental program
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Embodiment 1

[0024] Obtaining of internal reference gene sequence of embodiment 1 E. aeruginosa beetle

[0025] The method for obtaining the internal reference gene sequence of E. aeruginosa beetle comprises the following steps:

[0026] (1) Collection and rearing of A. aeruginosa beetles: adults were collected in the Laixi experimental field of Peanut Research Institute of Shandong Province, and raised in a breeding box covered with 20-30cm soil. The breeding box was placed in an artificial climate box, and the feeding condition was 26 ± 1 ℃, the photoperiod is 16L:8D, and the soil moisture content is 18%-20%.

[0027] (2) Obtain candidate internal reference gene sequences: actin-1 (SEQ ID NO:4), actin-2 (SEQ ID NO:5), GAPDH (SEQ ID NO:6), GPDH (SEQ ID NO:7), RPL3 (SEQ ID NO:3), RPL23 (SEQ ID NO:1), RPL49 (SEQ ID NO:8), RPS14 (SEQ ID NO:9), E2F2 (SEQ ID NO:10), ARF6 (SEQ ID NO: The sequence of 2) was obtained by transcriptome sequencing. The specific process was to use the lluminaHiSeq ...

Embodiment 2

[0034] The screening of embodiment 2 internal reference gene

[0035] The specific steps for the screening of internal reference genes stably expressed in different developmental stages and different tissue parts of E. aeruginosa are as follows:

[0036] Total RNA was extracted from eggs, 1st, 2nd, 3rd instar larvae, pupae, male and female adults, and antennae, head, thorax, abdomen, and legs of male and female adults according to the instructions of Trizol reagent (Invitrogen). The first strand of cDNA was synthesized according to the method of PrimeScript RT reagent kit with gDNAEraser kit, that is, RNA was reverse transcribed into cDNA.

[0037] Based on the nucleotide sequence (SEQ ID NO: 1-10) of each internal reference gene obtained in Example 1, utilize Primer Premier5.0 software to design and synthesize a pair of fluorescent quantitative specific primers for specific amplification respectively (Table 2 ), the specificity of the primers was determined by the single pea...

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Abstract

The invention provides an anomala corpulenta internal reference gene. The internal reference gene is ribosomal protein L23 (RPL23), an ADP ribosylation factor 6 (ARF6) and ribosomal protein L3 (RPL3),wherein the nucleotide sequence table of the RPL23 is as shown in SEQ ID NO.1, the nucleotide sequence table of the ARF6 is as shown in SEQ ID NO.2, and the nucleotide sequence table of the RPL3 is as shown in SEQ ID NO.3. The current situation that a reliable internal reference gene does not exist in an anomala corpulenta fluorescent quantitative PCR experiment is solved; and the screened internal reference gene is suitable for analyzing the expression level of a functional gene at different development stage or different tissue parts of the anomala corpulenta, and the reliability, the stability and the repeatability of the detection result can be improved.

Description

technical field [0001] The invention belongs to the technical field of internal reference genes stably expressed in insects in molecular biology, and specifically relates to an internal reference gene stably expressed in different developmental stages and different tissue parts of A. aeruginosa aeruginosa and its application. Background technique [0002] Anomalacorpulenta belongs to the phylum Arthropoda, class Insecta, order Coleoptera, and family Anomalacorpulenta. It is the third dominant species of scarabs in my country and is an important agricultural and forestry pest. Adults like to eat the leaves of apples, pears, peaches and other fruit trees and forest trees, as well as the leaves of crops such as peanuts and beans. The larvae feed on peanut pods and roots, potato roots and tubers, underground parts of wheat, beans, corn, vegetables, and saplings. When the number of insects is too large, they can also eat around the roots or bast parts of stems. The aeruginosa be...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/158C12Q2600/166C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 李晓曲明静鞠倩姜晓静杜龙焦坤彭娅萍石程仁李建文
Owner 青岛罗素生物技术有限公司
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