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Nervilia plicata is a rare southern Chinese herb. The study on the plant at molecular level is hardly seen. Real-time quantitative PCR (qRT-PCR) has been widely used in plant gene expression analysis, but there is a lack of research on it in N. plicata, which limits the progress of related work. Based on the previous researches, nine candidate reference genes, including Actin, GAPDH, TUA, UBC, UBQ, EF-1α, EF-1β, CYP and RPL, were screened out from the transcriptomic sequencing data. In order to select suitable reference gene in N. plicata, qRT-PCR technique was employed to detect the expression levels of the candidate reference genes in leaf, petiole and corm tissues of the plant. After analyzing the expression levels, the stability and comprehensive analysis of nine genes in different tissues, the most suitable reference gene was obtained. Finally, the most suitable reference gene was validated using NpDFR, Np3GT that related to anthocyanin synthesis. Results showed that the expression stability of EF-1 α and CYP was similar, with the best overall stability. When using either EF-1α or CYP, or the combination of them, as reference, the expression patterns of NpDFR and Np3GT in different tissues were consistent with the transcriptome sequencing results. This indicates that both EF-1 α and CYP can be used as reference genes in N. plicata for qRT-PCR. This study would provide a basis for further research on the molecular mechanism of genes related to the pharmacological effects of N. plicata.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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