《植物生理学报》 2018, 54(3): 509-517

菊叶薯蓣不同发育时期块茎内参基因的筛选

曾德福, 周建婵, 钟春梅^*, 谢君^*

华南农业大学林学与风景园林学院, 广东省高校生物质能源重点实验室, 农业部能源植物资源与利用重点实验室, 广州510642

通信作者：钟春梅；E-mail: xiejun@scau.edu.cn, zhongchunmei@scau.edu.cn

摘　要：

薯蓣皂苷元为重要的药物原料。为初步分析菊叶薯蓣(Dioscorea composita)块茎中薯蓣皂苷元合成关键基因的表达, 筛选稳定表达的内参基因至关重要。根据已建立的菊叶薯蓣转录组数据库和已报道的传统内参基因, 筛选出ubiquitin-conjugating enzyme E2 (UBC7)、MMS ZWEI homologue 3 (MMZ3)、tubulin beta-7 (TUB7)、actin 1 (ACT1)、glyceraldehyde-3-phosphate dehydrogenase C subunit 1 (GAPC1)、elongation factor 1-alpha (EF-1α2)、cyclophilin 5 (CYP5)、histone H2A 2 (H2A2)共8个候选内参基因, 利用实时荧光定量PCR (qPCR)技术, 结合GeNorm、NormFinder和BestKeeper三个统计学软件进行分析。结果表明, 在菊叶薯蓣块茎不同发育过程中, TUB7和H2A2表达最稳定, 为最佳内参基因。本研究为探究薯蓣皂苷元合成途径分子机理提供参考。

关键词：菊叶薯蓣; 内参基因; qPCR; GeNorm; NormFinder; BestKeeper

收稿：2017-09-11   修定：2018-01-10

资助：国家科技支撑计划项目(2015BAD15B03)。

Screening of reference genes in Dioscorea composita tubers of different development stages

ZENG De-Fu, ZHOU Jian-Chan, ZHONG Chun-Mei^*, XIE Jun^*

College of Forestry and Landscape Architecture, South China Agricultural University; Key Laboratory of Biomass Energy of Guangdong Regular Higher Education Institutions; Key Laboratory of Energy Plant Resources and Utilization, Ministry of Agriculture, Guangzhou, 510642, China

Corresponding author: ZHONG Chun-Mei; E-mail: xiejun@scau.edu.cn, zhongchunmei@scau.edu.cn

Abstract:

Dioscorea composita of the family Dioscoreaceae produces the pharmaceutically important compound diosgenin. In order to study the expression patterns of the key genes in diosgenin biosynthetic pathway in D. composita tubers, screening and validation of the steadily expressing reference genes are inevitable. We analyzed D. composite transcriptome and selected eight candidate genes such as ubiquitin-conjugating enzyme E2 (UBC7), MMS ZWEI homologue 3 (MMZ3), tubulin beta-7 (TUB7), actin 1 (ACT1), glyceraldehyde-3-phosphate dehydrogenase C subunit 1 (GAPC1), elongation factor 1-alpha (EF-1α2), cyclophilin 5 (CYP5) and histone H2A 2 (H2A2) for expression stability analysis. Statistical analysis of quantitative real-time PCR (qPCR) results using GeNorm, NormFinder and BestKeeper showed that the expression of TUB7 and H2A2 are the most stable in tubers, and thus can be used as reference genes for qPCR analysis in D. composita. In summay, our findings identified suitable reference genes for qPCR in D. composita and laid foundation for studying gene expression in diosgenin biosynthetic pathway.

Key words: Dioscorea composita; reference genes; qPCR; GeNorm; NormFinder; BestKeeper