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The Partial Sequence, Cloning Method and Application of Internal Reference Gene of Aphid argentina

A technology of wheat long tube aphid and cloning method, which is applied in the field of molecular biology and can solve the problems affecting the accuracy of quantitative results and the like

Active Publication Date: 2021-09-03
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • The Partial Sequence, Cloning Method and Application of Internal Reference Gene of Aphid argentina
  • The Partial Sequence, Cloning Method and Application of Internal Reference Gene of Aphid argentina
  • The Partial Sequence, Cloning Method and Application of Internal Reference Gene of Aphid argentina

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Cloning of the Aphid aphid SA-HEL gene, SA-RPL14 gene, SA-RPL11 gene or SA-28S gene

[0065] 1. Extraction of total RNA from Aphid arvensis:

[0066]Utilize TRizol (Amion, USA) to extract the total RNA of the tube aphid (Amion, USA), and the specific method is as follows: take a 30 mg sample of the tube aphid apteridae, fully grind it in liquid nitrogen, then quickly add 1 ml Trizol lysate, fully shake for 20 s, and rest 5-10 minutes; add 200 μL of chloroform, shake for 20s, stand still for 5 minutes, centrifuge at 12,000 rpm at 4°C for 15 minutes; take the supernatant, add it to a centrifuge tube containing 2.5 times the volume of absolute ethanol, mix it upside down, and stand still for 10 minutes, - Store in refrigerator at 80°C for 2-4 hours; take out the sample and centrifuge at 7500rpm at 4°C for 10min; discard the supernatant, add 1ml of 75% ethanol, wash the precipitate twice, centrifuge at 12,000rpm at 4°C for 10min; add 30μL Rnase-free water Dissolv...

Embodiment 2

[0083] Design and synthesis of embodiment 2 fluorescent quantitative PCR amplification primers

[0084] According to the sequence of the gene fragments cloned in the NCBI of Aphids agriculi and Example 1, the primers for the fluorescent quantitative PCR of the gene were designed. Use UNAFold online software (http: / / mfold.rna.albany.edu / ?q=mfold / DNA-Folding-Form) to analyze the secondary structure of the nucleic acid partial sequence of each gene. The software settings are as follows: melting temperature, 60°C; DNA sequence, linear; Na+concentration, 50mM; Mg 2+ concentration, 3mM; other options are the initial settings. After obtaining the site containing the stem-loop structure in each gene template sequence, use the NCBI-Primer-BLAST online software (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHome ) to design primers, the software settings are as follows: primer melting temperature, 57-63°C; primer GCcontent, 40-60%; PCR product size, 150-300bas...

Embodiment 3

[0093] Example 3 Fluorescent quantitative PCR detection of SA-HEL gene in the adult aphids with and without wings of the long tube aphid

[0094] 1. Extraction of total RNA from winged and non-winged adult aphids:

[0095] TRizol (Amion, USA) was used to extract the total RNAs of the adult aphids winged and aphidless, respectively, and the specific method was the same as the method for extracting the total RNA of the aphids in Example 1.

[0096] 2. Reverse transcription

[0097]Use TAKARA PrimeScript 1st Strand cDNA Synthesis Kit for reverse transcription, the specific operation steps are as follows: take 1 μg total RNA of Aphid sativa into a sterile PCR tube, add 1 μL Ligo dT Primer, 1 μL dNTPMixture, use RNase-removed sterilized deionized water Make up to 10 μL; mix well, place at 65°C, heat for 5 minutes, and cool quickly on ice; add 4 μL 5×PrimeScript Buffer, 0.5 μL RNase Inhibitor, 1.0 μL PrimeScript RTase, 4.5 μL RNase free H to the 10 μL reaction solution 2 O, carry ...

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Abstract

The invention discloses a partial sequence, a cloning method and an application of an internal reference gene of the elongated tube aphid, and belongs to the field of molecular biology, and specifically relates to internal reference genes SA‑HEL, SA‑RPL14, SA‑SA‑RPL11, SA -28S, its nucleotide sequence is shown in SEQ ID No.1, SEQ ID No.6, SEQ ID No.11, SEQ ID No.16 respectively. The nucleotide sequence of the internal reference gene of the aphid aphid obtained by the present invention can be used as an internal reference gene for the future research on the functional gene of the aphid or gene expression in different developmental stages, the gene expression in the body of the aphid and the aphid, or as an internal reference gene for different aphids. Density treatment, or as a solution of the pheromone E‑β‑farnesene, or as a useful method for research under insecticide or antibiotic treatment.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to the internal reference gene SA-HEL gene, SA-RPL14 gene, SA-SA-RPL11 gene or SA-28S gene partial sequence, cloning method and its use as an internal reference gene in RT - Application in qPCR. Background technique [0002] Sitobion avenae Fabricius belongs to Homoptera and Aphididae. It is one of the main pests of wheat crops in my country. , sorghum, corn, sugar cane and other gramineous crops, as well as bluegrass, okra, crabgrass, clubhead grass, foxtail, white sheep grass and other gramineous and sedge weeds. It has the characteristics of wide distribution, large number and strong fecundity, which affects the normal growth of wheat plants, leads to a serious decline in wheat yield, and causes huge losses to agricultural production. In general years, wheat production can be reduced by 5.1% to 16.5%, and in major occurrence years, wheat production can be r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12Q1/6806C12Q1/6888
CPCC07K14/43563C12Q1/6888C12Q2600/166C12Q2600/124C12Q2600/158C12Q1/6851C12Q1/686C12Q2521/107C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 朱勋高海峰李祥瑞张云慧阎维巍程登发李新安任智萌龚培盼王超魏长平杨超霞李亚萍殷新田
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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