Studies On The Expression Of Terpenoids And Flavonoids And Their Synthesis Related Genes In Incarvillea Younghusbandii From Different Altitudes

Incarvillea younghusbandii Sprague,a small-sized herb in genus Incarvillea Juss.(Bignoniaceae),is growing in sandy meadows and gravel habitats at an altitude of3600-5400 m on the Tibetan plateau,and has important medicinal and economic value as a traditional Tibetan medicinal plant.As a result,plant materials from three different altitude habitats(4100m,4600 m,and 5200m)were chosen for metabolomic and transcriptomic analyses,and the following results were mainly obtained:(1)In order to investigate the differential metabolite profile of I.younghusbandii at different altitudes and between different parts of the plant,a total of 849 metabolites were identified from leaves and roots at three different altitudes using extensive metabolomics techniques.The biological pathways involved in the different metabolites were analyzed and the biological functions involved in the different metabolites were clarified.The accumulation patterns of terpenoids and flavonoids were also focused on,and it was found that the content of metabolites in leaves was generally higher than that in roots.And the flavonoids naringenin(pos＿3300),mulberry furan M(pos＿6304),6"-O-acetylgenistein(neg＿1705),lanosterin7-glucuronide-(1->2)-glucuronide(neg＿3025),geranoside(neg＿6249),lignan(neg＿6691)and the terpenoid metabolite Nerolides(neg＿5740),α-curcumin(pos＿4481),and(S)-olive bitter glucosinolates(pos＿5803)were highest in the leaves at the highest altitude.(2)Transcriptomic sequencing analysis of leaves and roots of I.younghusbandii at three different altitudes using high-throughput sequencing technology showed that differential genes between leaves and roots were mainly involved in processes such as flavonoid and flavonol biosynthesis,phenylpropanoid biosynthesis,arginine and proline metabolism,steroid biosynthesis and amino acid sugar and nucleotide sugar metabolism.Differential genes between leaves at different altitudes were mainly involved in biological pathways such as phenylpropanoid biosynthesis,pantothenic acid and Co A biosynthesis,metabolism of nicotinic acid and nicotinamide and cofactor biosynthesis as well as flavonoid biosynthesis and terpenoid biosynthesis.Differential genes between roots at different altitudes were mainly involved in the pathways of biotin metabolism,linoleic acid metabolism phenylpropanoid biosynthesis,glycine,serine and threonine metabolism,fructose and mannose metabolism and nicotinic acid and nicotinamide metabolism.(3)To further investigate the biosynthetic pathways of important secondary metabolites in I.younghusbandii,this study focused on genes related to terpenoid and flavonoid biosynthetic pathways,and their expression patterns were analyzed in different altitudinal environments and between different parts of the plant to screen for differentially expressed genes.In the comparison between leaves and roots,most genes were expressed at higher levels in leaves than in roots,a result consistent with metabolomics data.And terpene synthase genes(TPS＿3,TPS＿4 and TPS＿20),which are located downstream of terpene metabolite synthesis,and genes related to anthocyanin synthesis in flavonoid synthesis(CHS＿3,CHI＿2,HCT＿15,ANS,F3H＿3,BZ1＿3 and FG2)were found to be significantly up-regulated in expression with increasing altitude.(4)A network map of gene and metabolite correlations in the terpenoid and flavonoid biosynthetic pathways of I.younghusbandii was constructed by correlation analysis of transcriptomic data with metabolomic data based on Pearson correlation(Pearson)results.A total of 15 Uingenes obtained in the terpenoid biosynthetic pathway were strongly correlated with 45 metabolites.Strong correlations were obtained between 9 Uingenes and metabolites in the flavonoid biosynthetic pathway,and several MYB transcription factors significantly associated with flavonoid metabolites and genes were also identified in the flavonoid biosynthetic pathway,among which the expression levels of Iy MYB＿113 and Iy MYB＿166 transcription factors were found to be significantly up-regulated with elevation.(5)The sequences of Iy MYB＿113 and Iy MYB＿166 transcription factors wereobtained by gene cloning.Iy MYB＿113 is 842 bp in length and encodes 280 amino acids;Iy MYB＿166 is 906 bp in length and encodes 301 amino acids.Conserved domain analysis showed that both Iy MYB＿113 and Iy MYB＿166 have MYB conserved structural domains and are 1R-MYB type transcription factors.Evolutionary analysis showed that Iy MYB＿113 has high homology similarity to PHL1,At PHR1 and PHL4 and Iy MYB＿166 to At MYB80,and it is speculated that Iy MYB＿113 and Iy MYB＿166 may also have similar functions.We performed extensive metabolomic assays on the active substances in the leaves and roots of I.younghusbandii at three different altitudes,which we combined with transcriptomic data to focus on terpenoid and flavonoid biosynthetic pathways,and we analyzed the distribution of metabolites and the expression patterns.These findings also provide a theoretical foundation for the development of additional medicinal parts of the I.younghusbandii,as well as data support for further research into the molecular mechanisms of I.younghusbandii adaptation to extreme environments at high altitudes.