Primer and method for detecting JHE gene transcription level of agasicles hygrophila
A technology of A. chinensis and gene transcription, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of affecting expression regulation and increasing expression levels, and shorten the response Time, intuitive, quick and specific effects
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Embodiment 1
[0058] Handling of Test Material
[0059] In the control CO 2 Concentration (420μL·L －1 ) and high CO 2 Concentration (750μL·L －1 ) in the artificial climate box to cultivate the hollow lotus, which is used to raise the male and female A. chinensis. Different CO 2 Nine female and male worms with different concentrations were collected into Eppendorf tubes, and divided into three biological replicates, with a total of 12 groups of samples. After the worm samples were collected, they were quickly fixed in liquid nitrogen and stored at -80°C for later use.
Embodiment 2
[0061] Primer design
[0062] (1) Primer design: According to the sequence of the JHE gene of A. rosacea obtained by transcriptome sequencing, DNAMAN software was used to design specific primers suitable for fluorescent quantitative PCR detection. The primer sequences are as follows:
[0063] JHE-F:5`-GCCTATGGTAACTGGTCACAA-3`;
[0064] JHE-R: 5`-GCGTTGGATTTCTGTATTTAGCA-3`.
[0065] The length of the fluorescent quantitative PCR product of JHE gene of A. rosacea was 109 bp, and the result of agarose gel electrophoresis showed that the amplified product had the same length and was a single band, which indicated that the designed primers had strong specificity. Suitable for real-time fluorescent quantitative PCR detection, agarose gel electrophoresis results such as figure 1 shown.
[0066] (2) At the same time, according to the sequence of the A. chinensis tubulin gene obtained by transcriptome sequencing, primers for the internal control of fluorescent quantitative PCR were...
Embodiment 3
[0071] Extraction of total RNA
[0072] Refer to Quanshijin's TransZol TM Extract the total RNA according to the instructions of the Up Plus RNA Kit kit, grind the worms into powder with liquid nitrogen, add 1ml TransZol TM Up, transfer to a 1.5 ml centrifuge tube, let stand at room temperature for 5 minutes, then add 200 μL chloroform / 1 ml TransZol TM Up, shake vigorously for 30 s, and incubate at room temperature for 3 min. After centrifugation at 10,000 g at 4°C for 15 minutes, transfer the upper aqueous phase (generally <80%) to a new centrifuge tube, add 1 / 3 volume of absolute ethanol, and gently invert to mix. Put the RNA spin column in a 2 ml collection tube, transfer all the mixture from the previous step into the spin column, centrifuge at 12000 g for 30-60 s, discard the mobile phase, and reuse the collection tube. Add 500 μLCB9, centrifuge at 12,000 g for 30 s at room temperature, discard the mobile phase, then add 500 μLCB9, centrifuge at 12,000 g for 30 s...
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