Rsearch On Relative Expression And Key Genes Associated Synthesis Pathways Of Terpenoids And Flavonoids In Dendrobium Officinale Flower

Dendrobium officinale Kimura et Migo is an endangered medicinal plant of Dendrobium in Orchidaceae,which is a typical arbuscular plant.The flower is light yellow,its scent is fresh,the taste is light and clear,the medicine is good at clear the lungs.The floral fragrance and color of plant flowers are determined by volatile terpenoids and flavonoids,respectively.However,the related genes and molecular regulation mechanism of floral flower color formation of D.officinale are not yet clear.In this experiment,two cultivars of D.officinale（Wanhu No.5 and Wanhu No.6）were used as materials,and HS-SPME-GC-MS and UPLC-MS were used to construct a metabolic profile of volatile terpenoids and flavonoids during the blooming period.Joint analysis with transcriptome data,mining of differentially expressed genes in the biosynthetic pathways of terpenoids and flavonoids in different varieties of Dendrobium flowers,prediction of transcription factors regulating the synthesis of terpenoids and flavonoids,and providing theories and experiments for further exploration of its floral fragrance and flower color formation in accordance with.The main results are as follows:（1）HS-SPME-GC-MS was used to construct the metabolic spectrum of volatile terpenoids in D.officinale flowers.7 monoterpenoids and 2 sesquiterpenoids were detected in Wanhu No.5;6 monoterpenoids and 4 sesquiterpenoids were detected in Wanhu No.6.The highest relative content was pinene in two cultivars of flowers.UPLC-MS was used to detect the flavonoids in D.officinale flower.The different metabolites of the two cultivars of D.officinale flowers were ononin,rutin and spinosin.There were some differences in volatile terpenoids and flavonoids between Wanhu No.5 and Wanhu No.6.（2）Two complete flowers of D.officinale（Wanhu No.5 and Wanhu No.6.）at full flowering stage were selected for transcriptome analysis by second generation high-throughput sequencing（NCBI Publication No.:prjna70321）.A total of 18650 DEGs were obtained,KEGG analysis showed that 253 genes were related to terpenoid biosynthesis pathway and secondary metabolism pathway.WGCNA analysis was performed using transcriptome data,and DEGs were divided into 10 modules.After combined analysis of metabonomics and transcriptomics,44 key transcription factors related to terpenoids and flavonoids biosynthesis were screened.（3）DobHLH is a large family of transcription factors in plants,which has many functions,including insect attack,anthocyanin production,terpene synthesis and different signaling pathways.According to the transcriptome and D.officinale genome data,89 b HLH genes were screened and divided into 18 subfamilies according to cluster analysis,and the DobHLH family was analyzed by bioinformatics.Seven b HLH transcription factors of MYC subfamily were obtained by clustering,including DobHLH32,DobHLH88,DobHLH87,DobHLH22,DobHLH1,DobHLH24 and DobHLH18.Combined with the results of WGCNA and screened to show that DobHLH22 may be involved in the regulation of Do TPS5.（4）Do TPS5,a monoterpene synthase gene,was isolated in the early laboratory.Its function is to catalyze the synthesis of linalool using GPP as the substrate.In order to verify the regulation of candidate transcription factors on terpenoid synthetase,the full length of Do TPS5_pro,DobHLH22 and Do MYB32 were cloned and linked to the corresponding yeast vector.The results of yeast one hybrid of Do TPS5_prowith DobHLH22 and Do MYB32 showed that DobHLH22 and Do MYB32 could bind to Do TPS5_pro,and DobHLH22 and Do MYB32could participate in the regulation of Do TPS5.