Yang Yang, Ye Bi-Huan, Song Qi-Yan, Chen You-Wu, Hu Chuan-Jiu, DU Guo-Jian, Liao Rong-Jun, Li Hai-Bo
Zhejiang Agriculture and Forestry University Hangzhou 311300, China.
Zhejiang Academy of Forestry Hangzhou 310023, China.
Zhongguo Zhong Yao Za Zhi. 2020 Dec;45(24):5967-5975. doi: 10.19540/j.cnki.cjcmm.20200927.101.
In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, β-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.
为了分析黄精块茎中甾体皂苷生物合成途径相关基因的表达，选择在不同发育阶段和非生物胁迫下稳定表达的内参基因非常重要。根据先前建立的黄精转录组数据库和已报道的植物内参基因，本研究通过实时荧光定量PCR（qPCR）系统分析了8个候选内参基因，包括组蛋白H2A、甘油醛-3-磷酸脱氢酶、肌动蛋白、β-微管蛋白、泛素结合酶-E2-10、延伸因子1-α异构体、18S rRNA和α-微管蛋白4在黄精块茎不同发育阶段及茉莉酸甲酯（MeJA）胁迫下的表达稳定性。利用GeNorm、NormFinder和BestKeeper软件对qPCR结果进行统计分析，结果表明泛素结合酶-E2-10和延伸因子1-α异构体在黄精块茎不同发育阶段及MeJA胁迫下的表达最为稳定。通过分析甾体皂苷生物合成途径中4个基因的表达，进一步验证了这两个内参基因。综上所述，泛素结合酶-E2-10和延伸因子1-α异构体可作为黄精qPCR分析中最合适的内参基因。本研究也为今后深入研究黄精甾体皂苷生物合成途径的分子机制奠定了基础。
