The Study Of Anthocyanin Biosynthesis Pathway In Magnolia Wufengensis Based On The High-throughput RNA-seq

Magnolia(M.)wufengensis is a woody plant of Magnoliaceae.It has been used in urban landscaping because of its excellent floral shape and color characteristics.In this study,we used transcriptome sequencing,high performance liquid chromatography(HPLC)pigmentation analysis,qRT-PCR expression analysis and model plant transgene analysis to explore the biochemical and genetic basis of anthocyanin biosynthesis pathway in M.wufengensis at different flower development stages,and to verify key regulate genes,so as to provide the basis for genetic improvement of M.wufengensis flower color.At the same time,through the establishment of EST-SSR database based on the transcriptome data and the development of molecular markers in conjunction with public databases,a comprehensive analysis and marker system of M.wufengensis was developed to provide technical support for the development of molecular assisted breeding of M.wufengensis.The main achievements of this study are as follows:1.Qualitative and quantitative analysis of the flower color of M wufengensis ’JH1’ at the five developmental stages(S1-S5)showed that anthocyanin glycosides were significantly up-regulated at the S2 stage and peaked at the S3 stage,and the accumulation of anthocyanin was attributed to the increase of anthocyanin content in single cell and the increase of erythrocyte number through epidermal cell observation.2.Five main anthocyanin glycosides were identified by qualitative analysis of anthocyanin glycosides in M.wufengensis by liquid chromatography-mass spectrometry(LC-MS),including three cyanin glycosides,one paeoniflorin glycoside and one delphinidin glycoside.Therefore,the anthocyanin glycoside synthesis pathway of M.wufengensis contains two pathways:cyanidin glycoside and delphinidin glycoside.3.Using Illumina high-throughput transcriptome sequencing technology,transcriptome depth sequencing was carried out for the whole flower of M.wufengensis at different developmental stages.The unigene genes were assembled by de novo method and annotated by Nr,SwissProt,GO and KEGG to obtain high-quality transcriptome database of M.wufengensis.4.Through differential expression analysis,the genetic basis of the physiological and biochemical processes of M.wufengensis at different developmental stages were revealed for the first time,and it was determined that the S2 stage was the key period for the development and flower color formation of M wufengensis.At the same time,two important expression patterns related to anthocyanin secondary metabolism were screened by trend cluster analysis.5.Emphasis is laid on the structural genes of anthocyanin biosynthesis pathway in M.wufengensis.The regulation process of key synthase coding genes in anthocyanin biosynthesis pathway in M.wufengensis during development was explained more completely.A large number of transcription factor coding genes,including bHLH and MYB families,are also discovered,which can further study gene interaction and comprehensively understand the genetic regulation of anthocyanin glycoside in M.wufengensis in the future.The obtained control network provides a bank of candidate genes for future research.6.The key regulate gene MwMYB1 related to anthocyanin biosynthesis in M.wufengensis was screened.The conservative domain and promoter regulatory elements of the gene were predicted by bioinformatic analysis.Combining with evolutionary tree comparison and subcellular localization test,the coding protein of the gene was identified as an R2R2-MYB transcription factor.7.The biological function of MwMYB1 was studied.The expression pattern of MwMYB1 in M.wufengensis was elucidated as follows:in tepals,it mainly expressed in S2 phase and lasted to S4;besides tepals it also expressed in carpels,young leaves and petioles of M wufengensis;phenotypic analysis of transgenic tobacco and Arabidopsis thaliana and quantitative analysis of related genes by qRT-PCR showed that MwMYB1 could promote anthocyanin biosynthesis and possibly participate in the light-dependent anthocyanin biosynthesis.The potential regulatory pathways of MwMYB1 were studied by functional analysis of MwMYB1 promoter.On the one hand,it was proved that the expression sites of MwMYB1 included calyx,carpel and leaf;on the other hand,it indicated that MwMYB1 might receive ABA signal regulation.8.EST-SSR molecular marker system of M wufengensis was constructed based on transcriptome database for the first time,and the basic characteristics of EST-SSR of M.wufengensis were analyzed.The genetic characteristics of five M wufengensis cultivars were analyzed by screening EST-SSRs related to anthocyanin synthesis,which confirmed the reliability of EST-SSR and reflected a good correlation between EST-SSR and phenotype.At the same time,EST-SSR screened by application in other plants of the same family proved that EST-SSR of M wufengensis had better cross-species versatility potential.9.Based on the database of Liriodendron chinensis,the SSR molecular table marker system for M.wufengensis was constructed.Based on the general primer database of SRAP,the SRAP molecular marker system for M wufengensis was constructed.Together with the EST-SSRs,this study formed a comprehensive molecular marker system for M.wufengensis.These results indicate that the key indicator of anthocyanin glycoside synthesis in M.wufengensis is stage S2,which is regulated by specific members of key genes.In this study,Illumnia sequencing platform was used for the first time to conduct high-throughput sequencing of M wufengensis,and the related sequences obtained by sequencing were further screened by qRT-PCR technology for large-scale expression pattern analysis and validation of key regulatory genes.Based on the above conclusions,the basic genetic and biochemical basis of the biosynthesis pathway of M wufengensis was preliminarily analyzed.At the same time,the EST-SSR molecular marker system of M.wufengensis was developed for the first time,and the comprehensive molecular identification system of M.wufengensis was constructed.This work provides valuable genetic information and effective tools for flower color improvement and molecular assisted breeding,and have important theoretical and practical significance.