Expression of the ANS, CHS and DFR genes involved in the biosynthesis of anthocyanins in Vaccinium floribundum Kunth from Ecuador, using RT-qPCR.

Vaccinium floribundum Kunth, a wild native species of berry in Ecuador, presents a lot of phenolic compounds, specifically anthocyanins; hence it is considered a natural nutraceutical due to all its nutritional properties. The comparison of the expression of genes is involved in the biosynthesis pathway of anthocyanin of several populations. The aim of the research was to analyze the expression levels of three genes involved in the biosynthesis of anthocyanin in this species collected in two areas of the province of Pichincha: Machachi population of the Mejía canton, with geographic coordinates 0 ° 31′04.8 ″ S 78 ° 37′07.4 ″ W and altitude 3200 meters above sea level, and Pintag population of the Quito cantón, with geographic coordinates 0 ° 24′00.0 ″ S 78 ° 24′00.0 ″ W and altitude 3000 meters above sea level. The gene expression analysis was performed using the quantitative polymerase chain reaction technique and reverse transcription (RT-qPCR). For the population of Machachi, the glyceraldehyde-3-phosphate dehydrogenase gene had an average concentration of 648.59 ng/µL, followed by the chalcone synthase gene with 143.71 ng/µL, then by the dihydroflavonol 4-reductase gene with 59.58 ng/µL and finally by the anthocyanin synthase gene with 39 ng/µL. For the population of Pintag, the glyceraldehyde-3-phosphate dehydrogenase gene has an average concentration of 667.32 ng/µL, followed by the chalcone synthase gene with 157.22 ng/µL, then by the dihydroflavonol 4-reductase gene with 60.42 ng/µL, and finally by the anthocyanin synthase gene with 44.40 ng/µL. Each gene has a similar expression level in both populations, but there are differences when comparing the expression level among genes. Many enzymes, structural genes, and regulatory elements have been observed as transcription factors involved in anthocyanin biosynthesis.

Study on characterization of chalcone synthase gene from \(\textit{Pueraria lobata}\) and \(\textit{Pueraria mirifica}\) in Vietnam

Two species of genus Pueraria ((Pueraria lobata (synonym: Pueraria montana var. lobata) and Pueraria mirifica (synonym: Pueraria candollei var. mirifica)) are traditional plants used in medicine since ancient times. These plants have been used and became commercially crucial indigenous medicinal plants. Currently, both roots and flowers of P. mirifica are used as a dietary supplement and functional food for women because of their rich source of phytoestrogen and nutrition. However, little information of genes on both species of Pueraria genus (P. lobata and P. mirifica) are known in Vietnam. The purpose of this research is to support more understanding about Chalcone synthase (CHS) genes by determining and sequence analyzing an encoding region of CHS genes that were isolated from P. lobata and P. mirifica. The full-length open reading frame (ORF) sequence CHS was identified with 1170 bp which encodes 389 amino acids by Sanger sequencing. The isolated CHS gene of P. lobata has no difference in sequence with CHS reported on GenBank (D10223.1), whereas a difference of 26 nucleotide positions in CHS sequence of P. mirifica compared with the published gene sequence (JQ409456.1) as consequential having 97.78% genetic similarity. The CHS genes sequence of P. lobata and P. mirifica are homologous with 98.4% because of having 19 nucleotide differences. Chalcone-stilbene synthase N-C terminal, PLN03173, CHS-like, BH0617, fabH are some important domains predicting the CHS genes. Especially, the family signature ‘GVLFGFGPGLTI’ motif of CHS gene as a part of the active-site scaffold contributes to decide the product of cyclization reactions performing the stereochemistry of cyclization which was also observed in P. lobata and P. mirifica, but it was not included for all members in Fabaceae family. With in sillico analysis, the P. lobata and P. mirifica CHS sequences have highly conserved regions to maintain their structure and function, so that it needs further studies to clarify these points.

Multi-tissue transcriptome analysis of two Begonia species reveals dynamic patterns of evolution in the chalcone synthase gene family

Begonia is an important horticultural plant group, as well as one of the most speciose Angiosperm genera, with over 2000 described species. Genus wide studies of genome size have shown that Begonia has a highly variable genome size, and analysis of paralog pairs has previously suggested that Begonia underwent a whole genome duplication. We address the contribution of gene duplication to the generation of diversity in Begonia using a multi-tissue RNA-seq approach. We chose to focus on chalcone synthase (CHS), a gene family having been shown to be involved in biotic and abiotic stress responses in other plant species, in particular its importance in maximising the use of variable light levels in tropical plants. We used RNA-seq to sample six tissues across two closely related but ecologically and morphologically divergent species, Begonia conchifolia and B. plebeja, yielding 17,012 and 19,969 annotated unigenes respectively. We identified the chalcone synthase gene family members in our Begonia study species, as well as in Hillebrandia sandwicensis, the monotypic sister genus to Begonia, Cucumis sativus, Arabidopsis thaliana, and Zea mays. Phylogenetic analysis suggested the CHS gene family has high duplicate turnover, all members of CHS identified in Begonia arising recently, after the divergence of Begonia and Cucumis. Expression profiles were similar within orthologous pairs, but we saw high inter-ortholog expression variation. Sequence analysis showed relaxed selective constraints on some ortholog pairs, with substitutions at conserved sites. Evidence of pseudogenisation and species specific duplication indicate that lineage specific differences are already beginning to accumulate since the divergence of our study species. We conclude that there is evidence for a role of gene duplication in generating diversity through sequence and expression divergence in Begonia.

Multi-Tissue Transcriptome Analysis of Two Begonia Species Reveals Dynamic Patterns of Expression Evolution In The Chalcone Synthase Gene Family

Begonia is an important horticultural plant, as well as one of the most speciose Angiosperm genera, with over 2000 described species. Genus wide studies of genome size have shown that Begonia has a highly variable genome size, and analysis of paralog pairs has previously suggested that Begonia underwent a whole genome duplication. We address the contribution of gene duplication to the generation of diversity in Begonia using a multi-tissue RNA-seq approach. We chose to focus on the chalcone synthase (CHS) gene family due to its role in biotic and abiotic stress response, and in particular its importance in maximising the use of variable light levels in tropical plants. We used RNA-seq to sample six tissues across two closely related but ecologically and morphologically divergent species, Begonia conchifolia and B. plebeja, yielding 17,012 and 19,969 annotated unigenes respectively. We identified the chalcone synthase gene family members in our Begonia study species, as well as in Hillebrandia sandwicensis, the monotypic sister genus to Begonia, Cucumis sativus, Arabidopsis thaliana, and Zea mays. Phylogenetic and expression analysis revealed the recent origin of CHS duplicates in Begonia, which showed both conserved and divergent expression profiles between duplicates. We conclude that there is evidence for a role of gene duplication in generating diversity through expression divergence in Begonia.

Identification of a novel anthocyanin synthesis pathway in the fungus Aspergillus sydowii H-1

Background Anthocyanins are common substances with many agro-food industrial applications. However, anthocyanins are generally considered to be found only in natural plants. Our previous study isolated and purified the fungus Aspergillus sydowii H-1, which can produce purple pigments during fermentation. To understand the characteristics of this strain, a transcriptomic and metabolomic comparative analysis was performed with A. sydowii H-1 from the second and eighth days of fermentation, which confer different pigment production. Results We found five anthocyanins with remarkably different production in A. sydowii H-1 on the eighth day of fermentation compared to the second day of fermentation. LC-MS/MS combined with other characteristics of anthocyanins suggested that the purple pigment contained anthocyanins. A total of 28 transcripts related to the anthocyanin biosynthesis pathway was identified in A. sydowii H-1, and almost all of the identified genes displayed high correlations with the metabolome. Among them, the chalcone synthase gene (CHS) and cinnamate-4-hydroxylase gene (C4H) were only found using the de novo assembly method. Interestingly, the best hits of these two genes belonged to plant species. Finally, we also identified 530 lncRNAs in our datasets, and among them, three lncRNAs targeted the genes related to anthocyanin biosynthesis via cis-regulation, which provided clues for understanding the underlying mechanism of anthocyanin production in fungi. Conclusion We first reported that anthocyanin can be produced in fungus, A. sydowii H-1. Totally, 31 candidate transcripts were identified involved in anthocyanin biosynthesis, in which CHS and C4H, known as the key genes in anthocyanin biosynthesis, were only found in strain H1, which indicated that these two genes may contribute to anthocyanins producing in H-1. This discovery expanded our knowledges of the biosynthesis of anthocyanins and provided a direction for the production of anthocyanin.

Identification of novel anthocyanins synthesis pathway from fungus, Aspergillus sydowii H-1

Background Anthocyanins are common substance in people's production and life. Usually it was used as a natural dye, more and more studies have shown that anthocyanins have potential preventive and / or therapeutic effects on human health, such as improving cardiovascular function and treating obesity. However, anthocyanins are generally thought to occur only in natural plants. The difficulty of mass production limits the application of anthocyanins in industry. Production of anthocyanins from microbial fermentation is not restricted by time, space and environmental conditions as compared with plants, which makes it more anxious to obtain anthocyanins from microorganisms. Here, we firstly reported a fungus, Aspergillus sydowii H-1which can produce anthocyanins under the specific fermentation, and analyzed its metabolome and transcriptome. Results All of the 31 gene loci related to anthocyanins synthesis pathway were identified in A. sydowii H-1, and almost all of those genes display high correlation with the data from metabolome. Among them, chalcone synthase gene (CHS) and cinnamate-4-hydroxylase gene (C4H) were only found using denovo assembly without reference, and interestingly, the best hits of these two genes all belong to plant species. Therefore, the plant-derived genes, CHS and C4H, may be one of the reasons why A. sydowii H-1 can produce anthocyanins. We also identified 530 lncRNAs in our datasets, and among them, three lncRNAs targeted to the genes related to anthocyanins biosynthesis via cis-regulation, which likewise provide clues for the underlying mechanism of anthocyanins’ production in the fungi. Conclusions In this study, we discovered a fungus, Aspergillus sydowii H-1, which can produce anthocyanins under the specific fermentation condition. It’s the first report that anthocyanins can be produced in non-plant species.