CYP82E5 gene mutant for reducing nicotine conversion rate and application thereof
A CYP82E5, nicotine conversion rate technology, applied in the field of genetic engineering, can solve problems such as reducing nicotine conversion rate
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0018] Creation of flue-cured tobacco mutant library
[0019] 1. EMS treatment of flue-cured tobacco seeds
[0020] Seeds of flue-cured tobacco (variety: Yunyan 87) were soaked in 50% commercially available bleach solution for 12 minutes, then quickly washed with deionized water, and soaked in deionized water for 12 hours. Discard deionized water and add an equal volume of 0.5% EMS (ethyl methanesulfonate) for 12 hours. Discard the treatment solution, add deionized water to wash 6-8 times, about 1 minute each time. The seeds were collected in a Buchner funnel and drained for later use.
[0021] 2. Field planting of M1 plants
[0022] After EMS treatment, the seeds are sown on floating trays, one seed per hole, and transplanted to the field after emergence, and managed with normal agronomic measures. After budding, single plant was bagged and harvested to obtain M2 seeds.
[0023] 3. Mutant Genomic DNA Extraction and Sample Mixing
[0024] Use the kit to extract genomic D...
Embodiment 2
[0028] CYP82E5 Gene Termination Mutant Screening
[0029] 1. Tilling primers
[0030] The CYP82E5 gene has two exons, and the mutants in the first exon region were screened by Tilling technology. According to the genome sequence of the target gene,
[0031] The forward primer is CYP82E5_F:5'-GGTAATTTTGTATTTATTATATTATGCG-3';
[0032] The reverse primer is CYP82E5_R: 5'-TCATCCTTAGTATTTAGATAATCTAATT-3'.
[0033] 2. PCR amplification conditions
[0034] The PCR reaction system is as follows: the total volume is 10 μL, including 1.0 μL of 20 ng / μL DNA sample, 1.0 μL of 10×PCR buffer, 0.8 μL of dNTPs, 0.16 μL of each primer, 0.1 μL of Taq DNase, and 6.78 μL of ddH2O.
[0035] The PCR reaction program is as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 30 seconds; Anneal at ℃ for 30 seconds, extend at 72℃ for 90 seconds, run 40 cycles; finally extend at 72℃ for 5 minutes. PCR amplification products can be stored at 4°C.
Embodiment 3
[0041] CYP82E5 gene termination mutation verification
[0042]1. Genomic DNA extraction and PCR amplification of M3 generation mutants
[0043] According to the Tilling screening results of the M2 generation plants, the seeds of a single plant with a mutation in the target region of the CYP82E5 gene (M3 generation, number 572) were selected and sown in seedling trays. The genomic DNA of seedling leaves was extracted by kit method. CYP82E5_F and CYP82E5_R primers were used to amplify the first exon region of CYP82E5 gene using genomic DNA as template. The PCR reaction system is as follows: the total volume is 25 μL, including 1.0 μL of 20 ng / μL DNA sample, 2.5 μL of 10×PCR buffer, 2 μL of dNTPs, 0.5 μL of each primer, 0.3 μL of Taq DNase, ddH 2 O 18.2 μL. The PCR reaction program is as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds (1°C drop per cycle), extension at 72°C for 90 seconds, and 30 cycles; ex...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More