RNA-Seq-based Expression Of Flavonoid Synthesis Genes And Transcription Factors In Scutellaria

Scutellaria viscidula Bunge(Scutellaria, Labiatae), a perennial herb, is used as Traditional Chinese Medicine for its dry root, where the main activity constituents are flavonoids (baicalin, baicalein, etc). S. viscidula has many bioactivities such as anti-cardiovascular disease, anti-cancer and anti-inflammation. Currently, little is known about the genome data and genetic background of most medicinal plants, leading to the uncertainty regarding the biosynthesis pathway and molecular mechanisms of active components. With the rapid development and perfection of Illumina high-throughput sequencing technology, it has become a new hotspot that a large number of gengmic data has been produced, which will reveal the secondary metabolism pathways of medicinal plants, as well as the molecular mechanism of cloning, expression and regulation of key enzyme genes.At present, studies of S. viscidula were limited to some fields such as the evaluation and utilization of germplasm resources and the construction of expression vectors. However, it remains unclear about the genetic basis of bioactive compounds of flavonoids (baicalin, baicalein, etc). Also, little is known about the structure and expression regulation of genes encoding the key synthetic enzymes. In this study, we used S. viscidula as materials to build the transcriptome database by RNA-seq. Based on this database, we mined several key genes involved in flavonoids synthesis and analyzed the gene structure and expression of regulatory transcription factors by fluorescence quantitative PCR. Combining with the dynamic change of total flavonoids content accumulation and gene expression, we further explored the biosynthetic pathway and molecular mechanism of the active component flavonoids in S. viscidula. The main results were as below:1. We used the S. viscidula as materials. After RNA extraction, the transcriptome was sequenced by Illumina HiSeq/MiSeq. Finally, we obtained 42310834 reads and 40052 Unigenes, with an average length of 882bp and 1577bp for Unigenes and N50, respectively. Transcriptome database of S. viscidula were performed Blast alignments with 6 protein databases (NR, KO, SwissProt, Pfam, GO, KOG) and nucleotide database NT. There were 24892 Unigenes (62.14% of all) annotated to S. viscidula databases. Comparing with the KOG database, S. viscidula was divided into 26 gene functional annotation. Using Blast and ESTScan prediction, we obtained 33367 CDS for S. viscidula. Collectively, the establishment of S. viscidula transcriptome database will help provide novel approaches and methods for breeding by genetic engineering and exploring the molecular mechanisms of active ingredients.2. We identified 8925 SSR loci in S. viscidula through SSR analysis. Dinucleotide repeats were determined as the main type (51.69%), followed by single nucleotide and trinucleotide repeats (29.03%,18.32%). The main motif type was AG/CT in dinucleotide repeats (40.78%). The SSR analysis will establish the foundation of mining functional genes and breeding by marker-assisted selection.3. Through analyzing the flavonoids synthesis pathways of S. viscidula, we annotated 136 and 41 genes to phenylpropanoid biosynthesis and flavonoid biosynthesis, respectively. There are 4,1,2,11 and 5 genes annotated in Chalcone synthase gene (CHS), Chalcone isomerase gene (CHI), Flavanone 3-hydroxylase gene (F3H), Phenylalanine ammonia-lyase gene (PAL),4-coumarate CoA ligase gene (4CL), respectively. We also identified 88 MYB transcription factors (MYB) sequence,34 basic helix-loop-helix (bHLH) sequence,10 WD40 repeat protein (WD40) transcription factors sequence related to flavonoid biosynthesis in the transcriptome databases.4. Through bioinformatics analysis with NCBI database, we screened and identified partial sequences of genes (CHI, CHS and F3H) and transcription factors (bHLH and MYB2) related to the biosynthesis of flavonoids in S. viscidula. Using these sequences as templates, we designed primers and detected the expression level of the above five genes in root, stem and leaf of three periods of S. viscidula by Quantitative real-time PCR. The results showed that these genes were differently expressed in root, stem and leaf of each periods. Specifically, CHS, F3H and CHI were highly expressed in July. In addition, MYB2 was down expressed in each periods, indicating possible regulations to CHS and CHI, which was positive to CHS while negative to CHI. There is no significant correlation between these regulations.5. We extracted total flavonoids from each parts of S. viscidula in three periods. Using baicalin as reference, we estimated the content of total flavonoids of root, stem and leaf of three periods by UV spectrophotometry. The results showed that the content in July was significantly higher than those in May and January. For different parts of S. viscidula, root has the highest content, followed by stem, and leaf has the lowest content of total flavonoids. Given the expression level of CHS?F3H and CHI, we concluded that these genes were highly expressed in July when the content of total flavonoids reached high level, indicating a possible positive correlation between gene expression level and flavonoids content.In this study, we obtained and analyzed the transcriptome data of S. viscidula as well as the key genes involved in the synthesis pathways of flavonoids. Furthermore, we conducted a preliminary analysis on the correlation between the gene expression in the synthesis pathways of flavonoids and total flavonoids. These results will further provide theoretical and practical references for increasing flavonoids content of S. viscidula by genetic engineering.