DOI: 10.57760/sciencedb.j00196.00009
title: Data set of CYP85A3 gene expression, root length and germination rate
title: CYP85A3基因表达量、根长、发芽率数据集

description: Sampling and treatment methodsThe roots, stems, new leaves at the top, old leaves at the bottom, buds and flowers of potato tissue culture seedlings and transplanted for 40 days were sampled for tissue expression analysis. The tissue culture seedlings after 15 days of tissue culture were put into the culture medium of BR (0, 50, 100 nmol L-1), and the root samples were taken at 0, 3, 6, 9, 12, and 24 hours after treatment. At the same time, the root length was counted after 15 days of treatment.The original seed tubers harvested in the substrate greenhouse were stored at 4 ℃ in the cold storage. After 40 days, the buds (cortex), cortex and pith were taken for tissue expression analysis. Soak C10 tuber BR seeds for 30 minutes, and then take the buds of terminal buds at 0, 3, 6, 9, 12, and 24 hours. The samples are frozen in liquid nitrogen and stored at – 80 ℃ for standby. The treated tubers are stored at 4 ℃ in the dark. Here we uniformly stipulate that the sprout length is more than 2 mm, and the sprout length is more than 80%, which is the complete sprout. After two weeks, the sprout rate is counted, and then every other week, 30 seeds for each treatment, 3 times repeated.1.6 qRT PCR determinationThe tissue expression and induced expression characteristics of StCYP85A3 were determined by Real-time PCR (Roche, Switzerland). The reaction system (20 µ L) includes 10 µ L Trans Start Top Green qPCR Super Mix (full gold, Beijing), 1.2 µ L cDNA template, 0.5 µ L (10 µ mol L-1) of PCR upstream and downstream primers, and 7.8 µ L dd H2O. QRT-PCR reaction procedure: 95 ℃ 5 s, 56 ℃ 10 s, 72 ℃ 10 s, 50 cycles. With EF1 𝛼 L as the internal reference gene, 2- ΔΔ The relative expression of genes was calculated by Ct method.1.7 Genetic transformation of ArabidopsisThe inflorescence of wild type Arabidopsis (col-0) and cyp85a2 mutant was soaked [16], and the seeds of T0 generation matured. The positive seedlings of T0 generation plants were screened by kana (50 mg L-1) resistance, and then the homozygous lines of T2 generation were screened [17]. After NptII genome identification, the expression of StCYP85A3 was detected by qRT PCR, and the lines with the highest expression were selected as StCYP85A3/col-0 and StCYP85A3/cyp85a2, respectively, and used for follow-up research. The seeds of StCYP85A3/col-0 and StCYP85A3/cyp85a2 strains were disinfected [18] and seeded on 1/2MS and 1/2MS medium containing 50 nmol L-1 BR. After being vernalized at 4 ℃ for 2 days, they were placed vertically in the incubator for dark culture for 2 days and then changed to normal culture. The germination rate of seedlings was measured at 0, 24, 36, 48, 60, 72, 84, 96 hours after vernalization. After 5 days of normal culture, Arabidopsis seedlings on blank (1/2MS) medium were transplanted to new 1/2MS medium and 1/2MS medium containing 50 nmol L-1BR, and their leaves and roots were sampled at 0, 3, 6, 9, 12, and 24 hours respectively, and the taproot length was measured after 5 days. Each treatment was set with 3 biological repeats, each containing 30 seeds.
description: Sampling and treatment methodsThe roots, stems, new leaves at the top, old leaves at the bottom, buds and flowers of potato tissue culture seedlings and transplanted for 40 days were sampled for tissue expression analysis. The tissue culture seedlings after 15 days of tissue culture were put into the culture medium of BR (0, 50, 100 nmol L-1), and the root samples were taken at 0, 3, 6, 9, 12, and 24 hours after treatment. At the same time, the root length was counted after 15 days of treatment.The original seed tubers harvested in the substrate greenhouse were stored at 4 ℃ in the cold storage. After 40 days, the buds (cortex), cortex and pith were taken for tissue expression analysis. Soak C10 tuber BR seeds for 30 minutes, and then take the buds of terminal buds at 0, 3, 6, 9, 12, and 24 hours. The samples are frozen in liquid nitrogen and stored at – 80 ℃ for standby. The treated tubers are stored at 4 ℃ in the dark. Here we uniformly stipulate that the sprout length is more than 2 mm, and the sprout length is more than 80%, which is the complete sprout. After two weeks, the sprout rate is counted, and then every other week, 30 seeds for each treatment, 3 times repeated.1.6 qRT PCR determinationThe tissue expression and induced expression characteristics of StCYP85A3 were determined by Real-time PCR (Roche, Switzerland). The reaction system (20 µ L) includes 10 µ L Trans Start Top Green qPCR Super Mix (full gold, Beijing), 1.2 µ L cDNA template, 0.5 µ L (10 µ mol L-1) of PCR upstream and downstream primers, and 7.8 µ L dd H2O. QRT-PCR reaction procedure: 95 ℃ 5 s, 56 ℃ 10 s, 72 ℃ 10 s, 50 cycles. With EF1 𝛼 L as the internal reference gene, 2- ΔΔ The relative expression of genes was calculated by Ct method.1.7 Genetic transformation of ArabidopsisThe inflorescence of wild type Arabidopsis (col-0) and cyp85a2 mutant was soaked [16], and the seeds of T0 generation matured. The positive seedlings of T0 generation plants were screened by kana (50 mg L-1) resistance, and then the homozygous lines of T2 generation were screened [17]. After NptII genome identification, the expression of StCYP85A3 was detected by qRT PCR, and the lines with the highest expression were selected as StCYP85A3/col-0 and StCYP85A3/cyp85a2, respectively, and used for follow-up research. The seeds of StCYP85A3/col-0 and StCYP85A3/cyp85a2 strains were disinfected [18] and seeded on 1/2MS and 1/2MS medium containing 50 nmol L-1 BR. After being vernalized at 4 ℃ for 2 days, they were placed vertically in the incubator for dark culture for 2 days and then changed to normal culture. The germination rate of seedlings was measured at 0, 24, 36, 48, 60, 72, 84, 96 hours after vernalization. After 5 days of normal culture, Arabidopsis seedlings on blank (1/2MS) medium were transplanted to new 1/2MS medium and 1/2MS medium containing 50 nmol L-1BR, and their leaves and roots were sampled at 0, 3, 6, 9, 12, and 24 hours respectively, and the taproot length was measured after 5 days. Each treatment was set with 3 biological repeats, each containing 30 seeds.