The Molecular Regulation Network Of Flavonoid Biosynthesis In Mirabilis Himalaica Induced By Low Temperature

Flavonoids are polyphenols mainly induced by various environmental factors such as hard light,ultraviolet radiation,low temperature and drought.Flavonoids play great roles both in plant development and stress adaptation.In this thesis,the molecular network of flavonoids synthesis of cold-acclimated calli in Mirabilis himalaica was revealed on the basis of RNA-seq transcriptome sequencing data.The main results are as follows:1.Differential expressed genes related to flavonoid synthesis.The accumulation of flavonoids gradually increased with cold acclimation in Mirabilis himalaica callus,and reached peaks on 9th day,which was 1.9 times that of the control.38 unigenes were identified related to regulation of flavonoids pathway based on transcriptome data and DEG analysis,and anthocyanins and lignin are two important branches of flavonoid biosynthesis in Mirabilis himalaica.The expressions of C4 H,4CL,F3’H,IFR,FLS,UGFT,MRP3,GST and lignin synthesis genes HCT,C3 H,CCo AOMT,CAD,CCR,PER were up-regulated after low temperature treatment,which was consistent with the increase of flavonoid synthesis under low temperature.2.By means of LS-MS technology,secondary metabolite of Mirabilis himalaica was analyzed during cold-acclimation.Ten metabolites are relatively high,the highest is corynoxine,the second is kaempferol,Cyanidin 3-glucoside Isoquercitrin,Rhodojaponin I,Astragalin,Isorhamnetin 3-glucoside,lutein,(-)-Epigallocatechin,and vinblastine is the third.3.Transcription factors related to secondary metabolite synthesis under low temperature.A total of 49 unigene of transcription factors were screened from the transcriptome Data of Mirabilis himalaica callus.According to the results of differential gene enrichment,differentially expressed genes were screened,including MYB(37),AP2-EREBP(9),and b HLH(6).Conserved domain,protein subcellular localization,and phylogenetic tree analysis were analyzed.The 30 Mh MYB proteins were divided into 15 subfamilies according to the classification method of 70 Arabidopsis family genes.The homologous genes involved in flavonoids biosynthesis mainly belong to the S2,S7 and S20 subfamilies.4.Clone and genetic transformation of Mh MYBThe CDS sequence of a MYB gene has been cloned from calli of Mirabilis himalaica by PCR technology,and named as Mh MYB6.Bioinformatics analysis showed that the Mh MYB6 sequence is645 bp,belonging to SANT gene family.Phylogenetic tree analysis showed that Mh MYB6 are highly homologous with At MYB11,At MYB12,and At MYB11.The prokaryotic expression vector p ET28a-Mh MYB6 was successfully transformed into E.coli and a 34 k Da protein was induced by IPTG.The transgenic E.coli strain showed higher resistance to freeze,salt,and osmotic stress.In summary,this study preliminarily clarify the metabolic pathway of flavonoids biosynthesis induced by low temperature in alpine plant Mirabilis himalaica,and provides important informations for the utilization of this medical plant.