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Reference genes of Metopolophium dirhodum and screening method and application thereof

A technology of internal reference gene and long tube aphid, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., and can solve problems affecting the accuracy of quantitative results, etc.

Inactive Publication Date: 2020-05-19
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

With the wide application of qRT-PCR technology, studies have found that, depending on the type of insect sample and experimental conditions, the expression level of housekeeping genes will change greatly under certain conditions. If you use it blindly, it may affect the final quantitative results. the accuracy of

Method used

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  • Reference genes of Metopolophium dirhodum and screening method and application thereof
  • Reference genes of Metopolophium dirhodum and screening method and application thereof
  • Reference genes of Metopolophium dirhodum and screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Selection of Candidate Internal Reference Genes for Aphid aphidica

[0053] 1. Extraction of total RNA from Aphid sativa:

[0054] Take 6 samples of adult aphids and put them into a clean 1.5 ml RNase-free centrifuge tube, grind them into fine powder in liquid nitrogen, add 1 ml Trizol solution; immediately shake with an oscillator for 1 min to quickly dissolve it in the lysate; room temperature Shake continuously during 10 min to fully lyse the parasites; add 200 ul of chloroform, shake quickly to make the solution fully mixed to milky white, let stand for 3 min, 4 ℃, 12 000 rpm, and centrifuge for 15 min; take 400 ul of supernatant until containing Place in a centrifuge tube with 1 ml of absolute ethanol for 5 min, freeze at -80°C, and freeze for 4 to 5 hours; take out the centrifuge tube with RNA precipitation from the freezer, centrifuge at 4°C, 12 000 rpm for 10 min; Discard the supernatant carefully, add 1 ml of 75% ethanol, gently blow up the precipita...

Embodiment 2

[0069] Example 2 Quantitative PCR detection of internal reference gene expression in different geographic populations of A.

[0070] 1. Extraction of total RNA from Aphid sativa:

[0071] Collected in Yinchuan, Ningxia (38°48'54.78''N, 106°30'27.93''E), Langfang, Hebei (39°8'9.8''N, 116°10'4.05''E), Guiyang, Guizhou (26° 0' 34.08''N, 106°35'4.35''E) Apterous adults of three different geographic populations of Aphid aphidalis, quick-frozen in liquid nitrogen, and stored in a -80 ℃ refrigerator. TRizol (TIANGEN, Beijing, China) was used to extract the total RNA of Aphid aphidis, and the specific method was the same as that in Example 1.

[0072] 2. Reverse transcription

[0073] Reverse transcription was performed using the FastKing gDNA Dispelling RT SuperMix cDNA Synthesis Kit, and the specific operation steps were the same as those in Example 1.

[0074] 3. Fluorescent quantitative PCR amplification primers

[0075] Fluorescent quantitative PCR amplification primers are t...

Embodiment 3

[0080] Example 3 Quantitative PCR detection of the expression of internal reference genes of Aphid sativa at different developmental stages

[0081] 1. Extraction of total RNA from Aphid sativa:

[0082] The selected test insect is Ningxia wheatless aphid, the feeding conditions are 20±1°C, humidity 60%-80%, light:darkness=16h:8h. Collect about 40 1st-instar nymphs, 30 2nd-instar nymphs, 20 3rd-instar nymphs, 20 4th-instar nymphs, and 20 wingless adults in each tube. The tubes were numbered and placed in liquid nitrogen for quick freezing, and stored in a -80°C refrigerator. TRizol (TIANGEN, Beijing, China) was used to extract the total RNA of Aphid aphidis, and the specific method was the same as that in Example 1.

[0083] 2. Reverse transcription

[0084] Reverse transcription was performed using the FastKing gDNA Dispelling RT SuperMix cDNA Synthesis Kit, and the specific operation steps were the same as those in Example 1.

[0085] 3. Fluorescent quantitative PCR ampl...

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Abstract

The invention discloses reference genes of Metopolophium dirhodum, and sequences, a screening method and application thereof, belongs to the field of molecular biology, and particularly relates to thereference genes Actin, GAPDH, NADH, AK, SDHB, RPL8, EF1A and RPL4 of the Metopolophium dirhodum. Nucleotide sequences of the reference genes are respectively shown as SEQ ID No.1, SEQ ID NO.4, SEQ IDNO.7, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.16, SEQ ID NO.22 and SEQ ID NO.25. The nucleotide sequences of the reference genes of the Metopolophium dirhodum can provide a useful method for research of gene expression of functional genes of the Metopolophium dirhodum or different geographical populations, development stages and body parts in the future, or for different-temperature treatment, or for starvation treatment, or for insecticide treatment, or for antibiotic treatment, or for research of gene expression of winged and winingless Metopolophium dirhodum.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to 10 housekeeping genes Actin, GAPDH, NADH, AK, SDHB, RPL8, 18S, EF1A, RPL4, and HSP68 of Aphid aphidica in different geographical populations and developmental stages , body part, temperature treatment, starvation treatment, insecticide treatment, antibiotic treatment, winged or wingless test conditions for the screening method and application of internal reference genes stably expressed. Background technique [0002] Wheat aphid is the main pest in wheat production in my country, and its species mainly include the wheat aphid[ Sitobion avenae (Fabricius)], Cereal aphid [ Rhopalosiphum padi (Linnaeus)], Aphid aphidus [ Metopolophium dirhodum (Walker)], two-fork aphid [ Schizaphisgraminum (Rondani)]. Among them, the tube aphid is an important pest of wheat, oats, rye and other gramineous crops worldwide (Du Yuzhou, Mao Yaxun. Study on the life table of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/11C12Q1/6888C12Q1/686
CPCC07K14/47C12Q1/686C12Q1/6888C12Q2563/107C12Q2545/114
Inventor 朱勋李新安张云慧李祥瑞王邴婷鞠建松龚培盼王超魏长平杨超霞程登发
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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