中国中药杂志

2020, v.45(24) 5967-5975

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多花黄精块茎发育和胁迫条件下qPCR内参基因的筛选与验证
Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress

杨阳;叶碧欢;宋其岩;陈友吾;胡传久;杜国坚;廖荣俊;李海波;
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;Zhejiang Academy of Forestry;Quzhou Forestry Technology Popularization Station;

摘要(Abstract):

为分析多花黄精块茎中甾体皂苷生物合成通路基因的表达,筛选出块茎不同发育期以及在胁迫条件下稳定表达的内参基因至关重要。根据业已建立的多花黄精转录组数据库和相关报道的植物内参基因,以不同发育期和茉莉酸甲酯(methyl jasmonate, MeJA)处理后的多花黄精块茎为实验材料,利用实时荧光定量PCR(qPCR)技术,对8个候选内参基因包括histone H2A(H2A2),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),ACTIN,β-tubulin,ubiquitin-conjugating enzyme E2 10(UBQ-E2-10),elongation factor 1-alpha isoform(EF-1α2),18S rRNA(18S),α-tubulin 4进行系统的筛选;利用GeNorm,NormFinder,BestKeeper 3个统计学软件对内参基因的稳定性进行分析;通过分析甾体皂苷生物合成通路中4个基因的表达,对内参基因的可靠性进行验证。结果显示,以UBQ-E2-10作为内参基因时,4个甾体皂苷合成相关基因在不同发育期块茎中的表达情况,以及在受MeJA胁迫处理后块茎中的表达情况与以EF-1α2作为内参基因时的表达情况具一致性,表明UBQ-E2-10和EF-1α2均可作为合适的qPCR内参基因用于多花黄精不同发育期和外界逆境胁迫下块茎的基因表达分析。该研究所筛选出的2个内参基因UBQ-E2-10和EF-1α2为后续探讨多花黄精甾体皂苷生物合成途径的分子机制奠定了基础。
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.

关键词(KeyWords): 多花黄精;发育;茉莉酸甲酯;内参基因;实时荧光定量PCR
Polygonatum cyrtonema;development;methyl jasmonate;reference gene;Real-time quantitative PCR

Abstract:

Keywords:

基金项目(Foundation): 浙江省中央财政林业科技推广项目(2017-TS04);; 浙江省衢州市科技攻关项目(2018-K45)

作者(Author): 杨阳;叶碧欢;宋其岩;陈友吾;胡传久;杜国坚;廖荣俊;李海波;
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;Zhejiang Academy of Forestry;Quzhou Forestry Technology Popularization Station;

Email:

DOI: 10.19540/j.cnki.cjcmm.20200927.101

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