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利用已获得的苹果Ty1-copia类逆转座子LTRs建立了苹果逆转座子间扩增多态性(IRAP)技术体系,对影响IRAP-PCR扩增结果的若干因子进行了研究。优化的反应体系包含:2.5μL10×buffer,50 ng模板DNA,2.5mmol.L-1Mg2+,0.2 mmol.L-1dNTPs,0.4μmol.L-1引物,1 UTaqDNA聚合酶,反应体积为25μL;优化的PCR扩增程序为:94℃2 min;94℃1 min,退火1 min,72℃2 min,循环35次;72℃延伸10 min;退火温度根据引物温度设定。该IRAP体系分别用于‘元帅’和‘富士’的短枝和着色芽变指纹图谱的分析,构建的指纹图谱可以作为芽变鉴定的依据,表明这些突变可能系逆转座子转座插入或逆转座子间重组所致。
The reverse transposon amplification polymorphism (IRAP) system of apple was established by using Tyr-copia retrotransposon LTRs, and some factors influencing the results of IRAP-PCR amplification were studied. The optimized reaction system consisted of 2.5μL 10 × buffer, 50ng template DNA, 2.5mmol.L-1Mg2 +, 0.2mmol.L-1dNTPs, 0.4μmol.L-1 primer and 1 UTaq DNA polymerase with a reaction volume of 25μL. The PCR procedure was as follows: 94 ℃ for 2 min, 94 ℃ for 1 min, annealing for 1 min, 72 ℃ for 2 min, cycling for 35 cycles and extension for 72 ℃ for 10 min. The annealing temperature was set according to the primer temperature. The IRAP system was applied to the analysis of the sprouting and staining fingerprints of ’Marshal’ and ’Fuji’ respectively. The fingerprinting can be used as the basis for bud mutation identification, indicating that these mutations may reverse the insertion or reversal of the transposon Rebuild between the seat caused.