The Discovery And Expression Analysis Of Key Genes For Flavonoid Synthesis In Astragalus Membranaceus Based On Transcriptome Sequencing

Scutellaria baicalensis Georgi (Scutellaria, Labiatae), a perennial herb, is one of the commonly used traditional Chinese medicine. Its air-dried root has pharmacological effects of clearing heat and detoxication, and flavonoid which was extracted from shows biological and pharmaceutical properties, including anti-inflammation, choleretic action, anti-oxidation and inhibiting HIV-RT viruses. However, there is little study on the regulatory process and regulation gene about flavonoid biosynthetic pathway. Especially, it had not been reported on the relationship between key gene expression and the accumulation of flavonoids. This study is to explore the genes of key enzyme in the flavonoids synthesis pathway based on transcriptome dataset of Scutellaria baicalensis and the relationship between the expression of key gene and accumulation of flavonoids. We preliminarily reveal the molecular mechanisms of medicinal components in Scutellaria baicalensis to lay the foundation to improve the flavonoids content by gene engineering.In this study, we used the Illumina high-thtoughput sequencing technology to analyze the transcriptome of Scutellaria baicalensis and discover key genes related to the flavonoid biosynthetic pathway. Then we used Real-time PCR to analysis the expression of gene (CHS, CHI, F3H, bHLH, MYB2). We test the content of flavonoids compounds in Scutellaria baicalensis by the ultraviolet spectrophotometer. Finally, we mainly analyzed the relationship between expression of key gene and accumulation of flavonoids. The main results are following contents:1. After Illumina running, we got 45,463,590 clean reads, which assembled into 40,485 unigenes. We performed Blast with the public databases (NT, NR, KO, swissprot, Pfam, GO, KOG), All unigenes were annotated in public database. Functional assignment based on Kyoto Encyclopedia of Genes and Genomes(KEGG) terms revealed 7 genes involved in flavonoid biosynthetic pathway, such as PAL, C4H, 4CL, CHS, CHI, F3H and FLS. These results greatly enrich the database of Scutellaria baicalensis gene and discover key genes related to the flavonoid biosynthetic pathway.2. Using ultraviolet spectrophotometer to test the content of flavonoids, we found that:(l)There are great discrepancies about the distribution of the content of flavonoid in Scutellaria baicalensis. The result shows that:leaf>root>stem. (2) There are great discrepancies about the accumulation of flavonoid in Scutellaria baicalensis. The content in root shows that:September>July>May; May>July> September in stem and July> May> September in leaf. It lays the foundation for the rule of the accumulation of flavonoids and also provides guidelines for the best time of harvest of Scutellaria baicalensis.3. We use Real-time PCR to analyze the expression of gene (CHS, CHI, F3H, MYB2, bHLH). We also analyze the content of flavonoids. It aims to reveal the molecular mechanisms of medicinal components. The results show that:(1) CHS and CHI gene expressed in various organs of Scutellaria baicalensis. In the root, the expression of CHS and CHI gene (September>July>May) and the content of flavonoids (September> July> May) is consistent. We speculate that the expression of CHI and CHS gene and the accumulation of flavonoids are positive correlative. (2) F3H gene expressed in various organs of Scutellaria baicalensis. In the root, the expression of F3H gene (May>July> September) and the content of flavonoids (September> July> May) is inconsistent. We speculate that the expression of F3H gene and the accumulation of flavonoids are negative correlative. (3)The transcription factor of MYB2, bHLH expressed in various organs of Scutellaria baicalensis. The expression of the transcription factor of MYB2 and bHLH and the distribution of flavonoids in various organ (leaf>root>stem) of Scutellaria baicalensis are similar. We speculate that they are positive correlative.From the results above, we get important data on the transcriptome dataset of Scutellaria baicalensis and the structure and composition of key gene in flavonoid biosynthetic pathway. It preliminarily reveal the relationship among key genes(CHS, CHI, F3H), transcription factors (bHLH, MYB2) and flavonoids. It also lays the foundation to improve the content of flavonoids by gene engineering.