显齿蛇葡萄实时荧光定量PCR内参基因的筛选与验证

发布时间：2018-01-24 21:01

本文关键词： 显齿蛇葡萄实时定量PCR 内参基因看家基因肌动蛋白基因 -磷酸甘油醛脱氢酶基因 S核糖体rRNA基因 α-微管蛋白基因 β-微管蛋白基因多聚泛素酶基因　出处：《中草药》2017年06期 　论文类型：期刊论文

【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。
[Abstract]:Objective to screen Ampelopsis grossedentata real-time quantitative PCRs (. Real-time fluorescence quantitative PCR. Methods A degenerate primer was designed and six candidate internal reference gene fragments, including actin gene (actin), were cloned from grape by RT-PCR. The gene of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ribosomal rRNA gene (18 S-rRNAs), 伪 -tubulin gene (伪 -Tubulin). 尾 -tubulin gene (尾 -Tubulin) and polyubiquianase gene (UBQN) were used to detect the six candidate internal reference genes in different tissues and organs of Vitis davidii by qRT-PCR. Stem tip. Expression of young leaves, mature leaves, old leaves, stems and roots, with the help of GE Norm. The expression stability of six internal reference genes was evaluated by Norm Finder and Best Keeper software. The expression of phenylalanine ammonia-lyase gene (Ag-PAL) was evaluated by using phenylalanine ammonia-lyase gene (Ag-PAL). Results 18 S-rRNA was used to verify the stability of the selected internal reference gene. The expression of GAPDH and Actin was stable and suitable for the study of gene expression in different tissues of Vitis davidii. These three bases were used to analyze the relative expression of Ag PAL gene. The results showed that the change trend of their expression in different tissues and organs was basically the same. Conclusion the suitable internal reference gene for qRT-PCR analysis of Vitis davidifolia can be used to lay a foundation for the further study of related gene expression.
【作者单位】：福建农林大学作物遗传育种与综合利用省部共建教育部重点实验室;福建农林大学农产品品质研究所;福建农林大学生命科学学院;
【基金】：国家科技支撑计划(2013BAD01B05)
【分类号】：R284
【正文快照】： 实时荧光定量PCR(real-time fluorescencequantitative PCR,q RT-PCR)是一种在传统PCR技术基础上发展起来的新型核酸定量技术,具有定量准确、灵敏度高、重复性好和高通量等优点,已被广泛应用于基因的表达和转录组分析等研究中[1-4]。在利用q RT-PCR技术进行相对定量表达分析时

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