摘要
目的筛选适用于显齿蛇葡萄Ampelopsis grossedentata实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)的内参基因。方法设计简并引物,采用RT-PCR从显齿蛇葡萄中克隆6个候选内参基因片段,包括肌动蛋白基因(Actin)、3-磷酸甘油醛脱氢酶基因(GAPDH)、18 S核糖体rRNA基因(18 S-rRNA)、α-微管蛋白基因(α-Tubulin)、β-微管蛋白基因(β-Tubulin)和多聚泛素酶基因(UBQ)。应用qRT-PCR技术检测这6个候选内参基因在显齿蛇葡萄不同组织器官中(茎尖、嫩叶、成熟叶、老叶、茎和根)的表达情况。借助Ge Norm、Norm Finder和Best Keeper 3种统计学软件综合评价6个内参基因的表达稳定性。通过苯丙氨酸解氨酶基因(Ag PAL)的表达分析对筛选出的内参基因进行稳定性验证。结果 18 S-rRNA、GAPDH和Actin的表达稳定性较好,适合作为显齿蛇葡萄不同组织基因表达研究的内参基因,以这3个基因为内参基因分析Ag PAL基因的相对表达量,结果发现它们在各组织器官中的表达变化趋势基本一致。结论确定显齿蛇葡萄qRT-PCR分析的合适内参基因,为后续相关基因表达研究奠定基础。
Objective To screen reference genes for real time quantitative PCR（qRT-PCR） research in Ampelopsis grossedentata. Methods On the basis of the conserved sequences among plant species, six candidate reference genes（including Actin, 18 S-rRNA, GAPDH, α-Tubulin, β-Tubulin, and UBQ） were cloned from A. grossedentata by RT-PCR in this study. The expression stability of each reference gene in different tissues（shoot tip, young leaf, mature leaf, old leaf, stem, and root） were analyzed by three softwares（Ge Norm, Norm Finder, and Best Keeper）, followed by validation of the expression pattern of Ag PAL by qRT-PCR. Results Actin, 18 S-rRNA, and GAPDH expressed most stably in all samples and were suitable for reference genes, which were further confirmed by the transcript level analysis result of Ag PAL in different tissues. Conclusion This is the first report on the screening and validation of reference genes for qRT-PCR in A. grossedentata, which benefits future studies on gene expression in this species.
出处
《中草药》
CAS
CSCD
北大核心
2017年第6期1192-1198,共7页
Chinese Traditional and Herbal Drugs
基金
国家科技支撑计划(2013BAD01B05)
