BACKGROUND Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. RESULTS Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. CONCLUSIONS Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

中文翻译：

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葛根变种的转录组分析。异黄酮和雌二醇生物合成中的基因发现用mirifica。

背景葛根（Pueraria candollei var）。mirifica是一种传统上用作复兴草药的泰国药用植物，被称为植物雌激素的丰富来源，其中包括异黄酮和高度雌激素的miroestrol和deoxymiroestrol。虽然这些活动成分在坎氏体育中。mirifica已有一段时间了，关于其生物合成基因的实际知识仍然未知。结果重新考虑了米罗雌酚的生物合成，并提出了从异黄酮黄豆苷元开始的最合理的机理。使用组合的坎氏毕赤酵母进行了从头转录组分析。幼叶，成熟叶，结节性皮层和切皮的块茎的丝裂组织。总共166 使用蛋白质数据库组装了923个重叠群，以进行功能注释，并作为一个文库，用于鉴定可能参与异黄酮类化合物和miroestrol生物合成的基因。来自四个单独的库的二十一个差异表达的基因被确定为参与这些生物合成途径的候选者，并且它们各自的表达通过定量实时逆转录聚合酶链反应得以验证。值得注意的是，LC-MS / MS生成的异黄酮和蜜雌酚谱与四种类型组织中异黄酮生物合成基因的表达水平呈正相关。此外，我们确定了R2R3 MYB转录因子，可能参与了P. candollei var中异黄酮生物合成的调控。mirifica。为了确认关键的异黄酮生物合成基因P. candollei var。的功能。在我们的库中鉴定出的苜蓿异黄酮合酶与本生烟草叶片中的拟南芥MYB12转录因子（AtMYB12）瞬时共表达。值得注意的是，这些蛋白的联合表达导致了异黄酮染料木黄酮的产生。结论我们的研究结果提供了令人信服的证据，证明转录组和代谢组的整合是一种强大的工具，可用于识别坎多利耶氏球菌中可能存在的异黄酮和丁雌酚生物合成中的生物合成基因和转录因子。mirifica。值得注意的是，这些蛋白的联合表达导致了异黄酮染料木黄酮的产生。结论我们的研究结果提供了令人信服的证据，证明转录组和代谢组的整合是一种强大的工具，可用于识别坎多利耶氏球菌中可能存在的异黄酮和丁雌酚生物合成中的生物合成基因和转录因子。mirifica。值得注意的是，这些蛋白的联合表达导致了异黄酮染料木黄酮的产生。结论我们的研究结果提供了令人信服的证据，证明转录组和代谢组的整合是一种强大的工具，可用于识别坎多利耶氏球菌中可能存在的异黄酮和丁雌酚生物合成中的生物合成基因和转录因子。mirifica。