Journal Article

Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol

Suntichaikamolkul, Nithiwat, Prombutara, Pinidphon, Kobtrakul, Khwanlada, Tantisuwanichkul, Kittitya, Zumsteg, Julie, Sirikantaramas, Supaart, Schaller, Hubert, Yamazaki, Mami, Wannachart, Siriporn, Saito, Kazuki, Vimolmangkang, Sornkanok, De-eknamkul, Wanchai

2019

Overview

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 ( At MYB12) 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 .

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Agriculture / Analysis / Annotations / Arabidopsis / Arabidopsis thaliana / Biochemistry, Molecular Biology / Biomedical and Life Sciences / Biosynthesis / Botanics / Criminal investigation / Cytochrome / Daidzein / DNA binding proteins / Enzymes / Estrogens / Flavonoids / Gene expression / Gene Expression Profiling / gene expression regulation / Genes / Genetic Association Studies / Genistein / Genomics / Genomics and evolution / Herbal medicine / High-Throughput Nucleotide Sequencing / Instrument industry (Equipment) / Isoflavones / Isoflavones - biosynthesis / Isoflavones - genetics / Isoflavonoids / Leaves / Life Sciences / Medicinal plants / Metabolism / Metabolites / metabolome / Miroestrol / Nicotiana benthamiana / Physiological aspects / Phytoestrogens / Phytoestrogens - metabolism / plant estrogens / Plant Sciences / Polymerase chain reaction / Proteins / Pueraria / Pueraria - genetics / Pueraria - metabolism / Pueraria candollei / Pueraria candollei var. mirifica / Research Article / reverse transcriptase polymerase chain reaction / Reverse transcription / Signal transduction / Steroids - biosynthesis / Transcription (Genetics) / Transcription factors / Transcriptome / transcriptomics / Tree Biology / Tubers / Vegetal Biology / White Kwao Krua / Xenoestrogens