Molecular detection method for Cry1Ac toxin non-recessive resistance cadherin cytomere deletion mutation of cotton bollworm
A technology of cadherin and deletion mutation, applied in the biological field, can solve problems such as difficulty in determining the diagnostic dosage, taking a long time, affecting the development and implementation of effective resistance management, and achieving the effect of saving labor and resources
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Embodiment 1
[0025] Step 1: extraction of genomic DNA from single cotton bollworm larvae or adults.
[0026] (1) Obtain the larvae of cotton bollworm confirmed to be resistant homozygous, resistant heterozygous and sensitive individuals through biological assay methods and genetic crossing with sensitive strains respectively.
[0027] (2) Extract larval genomic DNA using a commercially available DNA extraction kit. This method uses the AxyPrep Genomic DNA Mini Kit from Ai Sijin Biotechnology (Hangzhou) Co., Ltd.
[0028] (3) Take 30 mg of abdominal tissue of 3-4 instar larvae or adults of cotton bollworm, add 350 mL of 0.01mol / LPH7.4 phosphate buffer solution and 0.9 μL RNaseA (50 mg / mL) solution to a 1.5 mL centrifuge tube and grind evenly . Collect 350μL tissue homogenate in a 2mL centrifuge tube.
[0029] (4) Add 150 μL of lysate (Buffer C-L) and 20 μL of proteinase K (15 mg / mL) solution, vortex for 1 minute, centrifuge briefly, and place it in a 56-degree water bath for 10 minutes. ...
Embodiment 2
[0050] The following describes the preferred technical scheme of the present invention for the detection method of cadherin intracellular region deletion mutation gene frequency in the cotton bollworm population
[0051] Step 1: extraction of genomic DNA from single cotton bollworm larvae or adults.
[0052] (1) Collect cotton bollworm larvae directly from the field or use high-pressure mercury lamps to trap cotton bollworm adults, soak the obtained test insects in 95% ethanol and bring them back to the laboratory, and randomly select 100 cotton bollworm larvae or adult individuals for gene Frequency detection.
[0053] (2) Use a commercially available DNA extraction kit to extract genomic DNA from larvae or adults. This method uses the AxyPrep Genomic DNA Mini Kit from Ai Sijin Biotechnology (Hangzhou) Co., Ltd.
[0054] (3) Take 30 mg of abdominal tissue of 3-4 instar larvae or adults of cotton bollworm, add 350 mL of 0.01mol / LPH7.4 phosphate buffer solution and 0.9 μL RNa...
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