Method for detecting 6-pyruvoyl tetrahydropterin synthase PTS gene expression of Italian bee through fluorescent quantitative PCR (Polymerase Chain Reaction) technology

A technology of acetone tetrahydropterin and Italian bees, applied in the biological field, can solve the problems of no research reports on Hymenoptera insects, and achieve the effects of shortening experiment time, improving sensitivity, ensuring reliability and repeatability

Inactive Publication Date: 2018-08-10
SERICULTURAL RES INST ANHUI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

BH 4 Anabolic networks have been studied in Drosophila melanogaster and silkworm in Lepidoptera, but there are almost no research reports in Hymenoptera.

Method used

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  • Method for detecting 6-pyruvoyl tetrahydropterin synthase PTS gene expression of Italian bee through fluorescent quantitative PCR (Polymerase Chain Reaction) technology
  • Method for detecting 6-pyruvoyl tetrahydropterin synthase PTS gene expression of Italian bee through fluorescent quantitative PCR (Polymerase Chain Reaction) technology
  • Method for detecting 6-pyruvoyl tetrahydropterin synthase PTS gene expression of Italian bee through fluorescent quantitative PCR (Polymerase Chain Reaction) technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Obtaining the PTS Gene Sequence of Italian Apis mellifera

[0037] Open the Beebase (http: / / hymenopteragenome.org / beebase / ) database, enter the keyword PTS in the search bar, and find a result of PTS. Click PTS to get its Beebase database gene number GB51600. Click to enter the gene description page, select the link of the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, enter NCBI, and get the gene number LOC412015, the mRNA number of Ref-seq is XM_006571326.2, Protein The number is XP_006571389.1, that is, the CDS sequence of PTS such as (SEQ ID NO.4) and the amino acid sequence such as (SEQ ID NO.5) are obtained.

Embodiment 2

[0039] Extraction of Total RNA from Apis mellifera

[0040] Three species (three parallel samples in each group) of healthy Italian bees (worker bees, drones and queen bees) were selected to conduct the experiment of measuring the relative expression of PTS in different bees. An appropriate amount of healthy Italian bees of different species were selected and stored in -20°C freezer.

[0041] (1) Take 5 Italian worker bees and place them in a sterile mortar, pour liquid nitrogen into them and quickly grind them thoroughly with a grinding rod. Set for 5min.

[0042] (2) Add 200 μL of chloroform into the centrifuge tube, shake vigorously for 5-10 minutes, and let stand for 5 minutes.

[0043] (3) Centrifuge at 12,000 rpm for 10 min at 4°C, transfer the upper colorless aqueous phase into a new sterile centrifuge tube, add 500 μL of isopropanol, mix by inverting gently, let stand for 10 min, and centrifuge at 12,000 rpm for 10 min.

[0044] (4) Carefully pour off the liquid in ...

Embodiment 3

[0048] cDNA synthesis

[0049] The total RNA sequence of Apis mellifera (SEQ ID NO.3) described in Example 2 was used as a template. Take a sterilized 0.2mL centrifuge tube and add the following reaction system (20μL system):

[0050] (1) Add the following reagents to the nuclease-free PCR tube in ice bath: 800ng of RNA is reversed.

[0051]

[0052] (2) Gently mix and centrifuge for 3-5s. After incubating the reaction mixture at 65°C for 5 minutes, ice-bath for 2 minutes, and then centrifuge for 3-5s.

[0053] (3) Place the test tube in an ice bath, and then add the following reagents:

[0054] 5×RT Buffer 4.0ul

[0055] Thermo Scientific RiboLock RNase Inhibitor (20U) 0.5ul

[0056] RevertAid Premium Reverse Transcriptase (200U) 1.0ul

[0057] (4) Gently mix and centrifuge for 3-5s

[0058] (5) Carry out the reverse transcription reaction on the PCR instrument according to the following conditions

[0059] ①Incubate at 25°C for 10 minutes

[0060] ②cDNA synthesis ...

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Abstract

The invention discloses a method for detecting 6-pyruvoyl tetrahydropterin synthase PTS gene expression of Italian bee through a fluorescent quantitative PCR (Polymerase Chain Reaction) technology andparticularly relates to the technical field of biology. A real-time fluorescent RT-PCR detection method for PTS of the Italian bee is established, and the method comprises the following steps: (1) performing gene detection and acquisition by using primers; (2) extracting total RNA; (3) synthesizing cDNA; (4) real-time fluorescent PCR; (5) performing relative expression calculation of PTS genes ofworker bee, drone and queen bee in the Italian bee. Compared with the conventional PCR, the real-time fluorescent RT-PCR adopted in the invention has the advantages that fluorescence signals are collected by an automatic instrument in the real-time fluorescent PCR technology, the subjectivity of judgment by naked eyes is avoided, and the sensitivity is further improved. The real-time fluorescentPCR technology is in a totally closed reaction, PCR after-treatment is not needed, pollution is avoided, and the reliability and repeatability of the results are ensured. Moreover, electrophoresis, quantitative scanning and other subsequent complicated steps in the conventional PCR are eliminated, and the experimental time is greatly shortened.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to specific primers for detecting the expression of the PTS gene of Apis mellifera, and a method for detecting the expression of the PTS gene of Apis mellifera 6-pyruvyltetrahydropterin synthase by fluorescent quantitative PCR technology. Background technique: [0002] As an important economic insect, honeybees are widely distributed all over the world. There are about 70 million bee colonies in the world. They play an important role in crop pollination and have extremely high economic and ecological value. Bee products, such as honey, royal jelly, propolis, etc., are rare natural products to maintain human health, and have created considerable social and economic benefits. [0003] With the release of the sequencing results of the honeybee genome in 2006, the honeybee became the fourth insect whose genome sequence was determined after Drosophila, Anopheles mosquito, and silkworm, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6876
CPCC12Q1/6851C12Q1/6876C12Q2600/158C12Q2531/113C12Q2563/107
Inventor 舒蕊
Owner SERICULTURAL RES INST ANHUI ACADEMY OF AGRI SCI
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