摘要
为快速定量大麦贮藏蛋白基因B-hordein差异表达,利用SYBR Green I染料法测定转基因大麦和对照花后10 d籽粒B-hordein表达量,通过RT-PCR扩增B-hordein和内参基因actin片段,对PCR退火温度、引物浓度等进行优化,结合扩增曲线和熔解曲线分析。结果表明,转基因大麦B-hordein基因表达量较对照品种Golden Promise显著降低(P<0.05),B-hordein和actin扩增片段分别在(84.51±0.01)℃和(80±0.01)℃处显示特异性单峰,可成功建立转基因大麦B-hordein SYBR Green I实时定量PCR法。
In this study,a qRT-PCR system with SYBR Green I was established to characterise the variation in the expression of B-hordein gene in barley.The target gene B-hordein and the reference gene actin were amplified with RT-PCR in the grain of 10 DAP(day after pollination)of transgenic barley and control.PCR annealing temperature,primers concentration and other reaction factors was optimized,and also the amplification curve and melt curve was analyzed.The results showed that the expression level of B-hordein of transgenic barley was significantly lower than that of control(P<0.05).Peak of B-hordein and actin were(84.51±0.01)℃and(80±0.01)℃,respectively,and the real-time quantitative PCR method of B-hordein SYBR Green I in transgenic barley could be successfully established.
作者
李静雯
张正英
王立光
陈军
朱天地
LI Jingwen;ZHANG Zhenying;WANG Liguang;CHEN Jun;ZHU Tiandi(Institute of Biotechology,Gansu Academy of Agricultural Sciences,Lanzhou Gansu 730070,China;Institute of Crops,Gausu Academy of Agricultural Sciences,Lanzhou Gansu 730070,China)
出处
《甘肃农业科技》
2020年第11期11-16,共6页
Gansu Agricultural Science and Technology
基金
国家自然科学基金(31660391、31460350)。
