Method for detecting Chouioia cunea Yang Hsp83 gene expression by utilizing fluorescent RT-PCR technique
A technique of RT-PCR and Pseudomonas serrata, which is applied in the field of detecting Hsp83 gene expression of Pseudomonas serrata using fluorescent RT-PCR technology, and achieves the effects of improving sensitivity, shortening experiment time, and ensuring reliability and repeatability
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Embodiment 1
[0046] Obtaining the Hsp83 Gene Sequence of Leptospermus zhouii
[0047] The experimental materials were obtained from the indoor subculture of the white moth A. zhouzii (cultivation conditions: in an artificial climate box (PQX-350H), temperature 25°C, relative humidity 60%-80%, no light and dark). The antennae of the female B. cerevisiae within 24 hours after eclosion were cut out under a dissecting microscope, and immediately soaked in RNAlater (Ambion, AM7020); the antennae of every 200 females of A. spp. In the centrifuge tube, a total of 8 tubes of samples were collected and stored at -20°C. After the samples were prepared, they were sent to Huada Gene Technology Service Co., Ltd. (http: / / www.genomics.cn / index) for transcriptome sequencing.
[0048] The high-throughput sequencing platform IlluminaHiSeq? 2000 was used to perform transcriptome sequencing on the antennae samples of A. chowii. Trinity software was used to cluster each read sequence fragment and then spliced ...
Embodiment 2
[0050] Extraction of total RNA
[0051] Select about 30 white moths, Chow's gnats, for different temperature treatments. The high temperature treatment (1h) is 28°C, 32°C, 36°C, 40°C and 42°C; ℃, -3℃, -7℃. The processed product was used immediately or stored frozen at -20°C.
[0052] (1) Take about 30 of the white moth Chow's gnats into a 1.5mL sterile centrifuge tube, pour liquid nitrogen into it and grind it quickly with a grinding rod. Add 600 μL Trizon solution to the centrifuge tube and let it stand for 5 minutes.
[0053] (2) Add 200 μL of chloroform into the centrifuge tube, shake vigorously for 15 seconds, and let stand for 3 minutes.
[0054] (3) Centrifuge at 12,000 rpm for 15 min at 4°C, transfer the upper colorless aqueous phase into a new sterile centrifuge tube, add 400 μL of isopropanol, place at -20°C for 20 min, and centrifuge at 12,000 rpm for 10 min.
[0055] (4) Carefully pour off the liquid in the tube, save the precipitate, and add 600 μL of 75% ethan...
Embodiment 3
[0059] cDNA synthesis
[0060] The total RNA of B. chowii described in Example 2 was used as a template. Take a sterilized 0.2mL centrifuge tube and add the following reaction system (20μL system): 5μL of TotalRNA, 1μL of orRandomPrimer (N9) (0.1μg / μL), 10μL of 2×TSReactionMix, 1μL of TransScript?RT / RIEnzymeMix, 3μL of RNase-freeWater, and mix well.
[0061] The reaction conditions were 10 minutes at 25°C in a water bath, 30 minutes at 42°C in a water bath, and 5 minutes at a high temperature at 85°C to inactivate the TransScript®RT retained in the centrifuge tube. The product was used immediately or stored frozen at -20°C.
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