茉莉花实时荧光定量PCR内参基因的筛选与验证

doi:10.7668/hbnxb.20191401

摘要/Abstract

摘要： 为筛选适合茉莉花的内参基因，以5种组织（根、茎、嫩叶、成熟叶、花）和4个发育阶段的花（幼蕾期、膨大花蕾期、最长花蕾期和初绽期）为试验材料，选择较常见的8个候选内参基因进行引物特异性分析，结果显示，8个内参基因均扩增出单一条带，溶解曲线均只有明显的单一峰。采用qRT-PCR技术分析8个内参基因在不同样品中的表达量，结果显示，内参基因在不同样品中的表达量存在差异，各基因在花中的表达量均显著低于其他样品。利用geNorm、NormFinder和BestKeeper软件对基因的表达稳定性进行评价，并通过RefFinder对表达稳定性进行综合分析，结果表明，适用于不同组织（有花组）的最优内参基因为SAND和UPL7；适用于不同组织（无花组）的最优内参基因为UPL7和GAPDH；而适宜不同发育阶段花的最优内参基因为Actin和EF1α。进一步利用JsDXS和JsPAL2对筛选的内参基因进行验证，结果显示，以稳定性好的Actin、EF1α及Actin+EF1α<组合为参照时，JsDXS和JsPAL2在花发育不同阶段的表达水平的变化趋势基本一致，而用最不稳定的PP2A为参照时，花发育不同阶段的表达水平的变化趋势不一致。总之，本研究对茉莉花内参基因进行了筛选，不同组织（有花组）的最优内参基因为SAND和UPL7；不同组织（无花组）的最优内参基因为UPL7和GAPDH；花发育不同阶段的最优内参基因为Actin和EF1α。

关键词: 茉莉花, qRT-PCR, 内参基因, 表达稳定性

Abstract: To screen suitable reference genes for jasmine, five tissues (root, stem, young leaf, matured leaf and flower) and four developmental stages of flowers (young floral bud, swollen floral bud, the longest floral bud and initial opening flower) of jasmine were used as experimental materials. The eight common candidate reference genes were selected for primer specificity analysis, and the results showed that all the eight genes amplified a single band, and the melting curve had only a single obvious peak. The expression levels of eight reference genes in different samples were analyzed by qRT-PCR. The results showed that the expression levels of eight candidate reference genes were different in the samples, and the expression level of each gene in flower was significantly lower than that of other samples. The stability of these genes was evaluated by software including geNorm, NormFinder and BestKeeper, and then the expression stability of them was comprehensively analyzed by software RefFinder. The results indicated that the best reference genes for different tissues with flowers were SAND and UPL7, for different tissues without flowers were UPL7 and GAPDH, and for different developmental stages of flowers were Actin and EF1α. Furthermore, the selected internal reference gene was verified by the target genes JsDXS and JsPAL2. The results showed that the expression levels of JsDXS and JsPAL2 presented the same trend at different stages when used Actin, EF1α and Actin+EF1α combination as references, however, the expression level trends were inconsistent when used the unstable PP2A as reference. In summary, this study we screened the reference genes for jasmine, and the optimal reference genes for different tissues including flowers were SAND and UPL7, but the most stable reference genes in different tissues without flowers were UPL7 and GAPDH. Simultaneously, Actin and EF1α were the most suitable genes for different developmental stages of flowers.

Key words: Jasmine, qRT-PCR, Reference gene, Expression stability

中图分类号:

S685.160.1

齐香玉, 陈双双, 冯景, 王华娣, 邓衍明. 茉莉花实时荧光定量PCR内参基因的筛选与验证[J]. 华北农学报, 2020, 35(6): 22-30. doi: 10.7668/hbnxb.20191401.

QI Xiangyu, CHEN Shuangshuang, FENG Jing, WANG Huadi, DENG Yanming. Selection and Validation of Candidate Reference Genes for Quantitative Real-time PCR in Jasminum sambac Aiton[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(6): 22-30. doi: 10.7668/hbnxb.20191401.

http://www.hbnxb.net/CN/Y2020/V35/I6/22

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