Reference gene for stable expression of red wheat blossom midges in different growth periods and application of reference gene

[0024] Embodiment 1: the preparation of sample

[0025] Take the insect soil with midges in spring, select and pick overwintering larvae of midges, and place the picked overwintering larvae in an incubator to raise them at a temperature of 20±1°C, a humidity of 70±5%, and a photoperiod of 14 hours: 10h (this time ratio is the time ratio of light and darkness), respectively get the samples of five different developmental stages of wheat red midge larvae, early pupae, hind pupae, male adults and female adults in the raising process, each sample that will choose is carried out respectively. Quick-frozen in liquid nitrogen and stored at -80°C for later use.

[0026] Embodiment 2: the extraction of sample RNA

[0027] Take 50-100 mg of each of the sample tissues of the five different developmental stages of the red midge prepared in Example 1, add 0.5-1 mL of liquid nitrogen to each of the sample tissues, and then add 1000 μL of TRIZOL reagent for grinding (first add 200 μL of TRIZOL reagent in the sample tissue, use the grinding rod matched with the centrifuge tube to grind until no obvious tissue can be seen, then add 800 μL TRIZOL reagent and shake well), after grinding, place it at room temperature for 5 minutes; then add 200 μL each Shake well with chloroform, shake well and place at room temperature for 2 to 3 minutes; place them in a refrigerated centrifuge and centrifuge at 4°C and 12,000g for 15 minutes; remove the supernatant, and then add 500 μL of isopropanol Shake well, shake well, place at room temperature for 10min, centrifuge at 4°C and 12000g for 10min respectively, remove the supernatant again, then add 1000μL of 75%

[0028] Example 3: reverse transcription

[0029] (1) DNA removal:

[0030] Take 5 μl of the RNA extracted in Example 2 (200ng / μl) and put it on ice, then add 5×gDNA EraserBuffer 2.0μl, gDNA Eraser 1.0μl and RNase Free dH 2 O 2 μl; after mixing evenly, react at 42°C for 2 min to obtain a DNA-depleted RNA solution, which is placed on ice for later use.

[0031] (2) Reverse transcription reaction:

[0032] The reaction system for reverse transcription is: 4 μL of 5×Prime Script Buffer 2 (for Real Time), 1 μL of Prime Script RT Enzyme Mix I, 1 μL of RT Primer Mix, 10 μL of RNA solution for removing DNA, and RNase Free dH 2O 4 μL, so that the total amount of the reaction system is 20 μL (the reaction system is prepared on ice); reverse transcription reaction is carried out after mixing various reaction solutions in the reaction system, the reaction conditions are 37 ° C, 15 min, and then 85℃, 5sec, 4℃, 10min;

[0033] cDNA samples were obtained after reverse transcription, and 1