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实时荧光定量PCR已广泛用于基因表达的分析,适当的内参基因选择是获得准确分析结果的关键。在大豆(Glycinemax)分子生物学研究中,逆境响应基因和microRNA(miRNA)表达的内参辅助检测基因均有哪些目前尚不清楚。该研究选用不同盐梯度和时间点组合处理的大豆根组织为材料,对已报道的其它条件下表达相对稳定的内参基因(ACT、ACT2/7、CYP2、ELF1A、ELF1B、F-Box、TUA和UBC2)以及miRNA内参基因(U6、miR1515a、miR1520c、miR1520d、miR171a和miR171b)的表达情况进行了全面检测;并采用?-Ct、Bestkeeper、NormFinder和Genorm四种方法对检测结果进行了综合分析,发现ELF1B和CYP2适合作为大豆根系盐胁迫响应基因研究的内参基因,miR1515a和U6适合作为盐胁迫下大豆根组织miRNA研究的内参。上述研究结果为大豆盐胁迫响应基因和miRNA表达及其进一步的功能研究奠定了基础。
Real-time fluorescence quantitative PCR has been widely used for gene expression analysis, and proper selection of internal reference genes is the key to obtaining accurate analysis results. In the Glycine max molecular biology study, it is not clear which genes are responsible for the expression of the adversity-responsive genes and microRNAs (miRNAs). In this study, soybean root tissues treated with different salt gradient and time-point combinations were used as materials. The results showed that the expression of the endogenous reference genes (ACT, ACT2 / 7, CYP2, ELF1A, ELF1B, F-Box, TUA and UBC2) and the expression of miRNA internal reference genes (U6, miR1515a, miR1520c, miR1520d, miR171a and miR171b). The results of the detection were comprehensively analyzed using the methods of? -Ct, Bestkeeper, NormFinder and Genorm ELF1B and CYP2 are suitable as internal reference genes for soybean root stress response genes, miR1515a and U6 are suitable as internal reference for the study of soybean root RNAi under salt stress. The above results laid the foundation for the study of salt-responsive gene and miRNA expression in soybean and their further functional studies.