The Study Of Anthocyanidins Biosynthesis Pathway In Senecio Cruentus Based On The High-throughput RNA Sequencing Technique

Cineraria (Senecio cruentus Masson ex L’Herit; Pericallis cruentia L’Herit) is a perennial herb plant within the Asteraceae family, which possesses ample flower colors and distinct color groups. In this study, Senecio cruentus’Jester’series including blue, carmine, pink and white color groups were selected as materials. By the methods of transcriptome sequencing, qPCR expression analysis and HPLC pigment analysis, we investigated the regulation mechanism of different anthocyanidins biosynthesis pathways which will provide reference for the study of color modification of ornamentals based on branch regulation. The main results we obtained are as follows.1. The analysis of pigment in Senecio cruentus cultivars’Xiaochou’series showed that branch pathway of delphinidin mainly existed in the blue group and pelargonidin in the pink group, both of which existed in the cyanidin pathway simultaneously, while branch pathway of cyanidin mainly existed in carmine group, which possesses branch pathway of delphinidin simultaneously. The accumulation pattern of different branch pathway outcomes at different developmental stages showed a trend of increased first and then decreased. Few anthocyanidins existed but some anthoxanthin existed in the white group, which could be considered as an ideal contrast material. According to the results of this study, we believed that Senecio cruentus is an important material for the study of the synthesis and branch regulation mechanism of anthocyanidins.2. We obtained52,879,884pieces of high-quality short sequences (90bp) using the transcriptome sequencing of ray flowers of the blue cineraria on different developmental stages by the Illumina/Solexa high-throughput transcriptome sequencing technique. Moreover,57,818pieces unigenes with an average length of708bp (N501074bp) were obtained after the de novo assembly of short reads by Trinity software. Accordingly, a high-quality and comprehensive transcriptome database of blue cineraria was constructed successfully.3. The CDS regions and orientation of sequences of43,367pieces of unigenes (75%of all unigenes) were certificated by the Nr annotation. The result of GO term classification and KEGG pathway enrichment indicated that those genes related to the process of metabolize, cell physiology, stimulus response and biological regulation were expressed massively, and those related to cellular metabolism, secondary metabolism and signal transduction of plant hormone played key roles in the regulation process. We selected some key genes and transcription factors related to the synthetize and decoration of anthocyanidins and transporter coding gene after the further analysis, and found that most of these genes belongs to multigene family.4. We screened out the reference genes which expressed stalely in the ray flowers of cineraria. The expression of SAND and Actin were very stable on different developmental stages of ray flowers of the pink and blue cineraria, while good performances of TIP41and Actin were appeared in the white group, and PP2A and Actin in the red group, which indicated that the combination of SAND, Actin and PP2A was much better for the gene expression analysis on different developmental stages of4different color groups.5. The analysis of expression patterns of the different members of11anthocyanidins-related gene families in ray flowers of4color groups and5inflorenscence developmental stages selected from the transcriptome database by qPCR method showed that different expression patterns appeared in different members, and some certain members participated in the progress of anthocyanidins synthesis. Those certain members regulated the key genes of different branch pathways were further screened out: ScF3’5’H and ScGT2gene only expressed in those samples possessed branch pathway of delphinidin; ScF3’H1gene promoted cyanidin branch pathway in the carmine cinerarias; ScF3’5’H and ScF3’H were no expression and low expression respectively in those samples possessed branch pathway of pelargonidin. The analysis of pigments and gene expression showed that the amount of expression and outcome were positive correlated.6. Through the qPCR expression analysis, we screened out coding genes ScBHLH17which regulates the transcription factors of ScF3Hl, ScDFR1and ScANS.7. We isolated10members belong to GST family, and confirmed that ScGST3is a relevant transporter related to anthocyanidins through the analysis of expression patterns.The above results indicated that the different branch pathways of anthocyanidins are regulated by key genes of certain members. We firstly sequenced cineraria by the Illumina/Solexa high-throughput transcriptome sequencing technique and obtained relevant sequences, and further screened out key regulating genes and coding genes of anthocyanidins transporter by qPCR technique. Based on the above conclusions, we preliminary analysis of the competition mechanism of the different branch of anthocyanidins biosysthesis pathway in Senecio cruentus, which provide references and gene resource for color modification of ornamentals, especially for the breeding of blue-flower species.