菊叶薯蓣不同发育时期块茎内参基因的筛选

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

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 H2A2(H2A2)共8个候选内参基因,利用实时荧光定量PCR(qPCR)技术,结合Ge Norm、Norm Finder和Best Keeper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。

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 H2 A 2（H2 A2） for expression stability analysis. Statistical analysis of quantitative real-time PCR（q PCR） results using Ge Norm, Norm Finder and Best Keeper showed that the expression of TUB7 and H2A2 are the most stable in tubers, and thus can be used as reference genes for q PCR analysis in D. composita. In summay, our findings identified suitable reference genes for q PCR in D. composita and laid foundation for studying gene expression in diosgenin biosynthetic pathway.