| Flavonoids are polyphenols mainly induced by various environmental factors such as hard light,ultraviolet radiation,low temperature and droughts.It plays great roles both in plant development and stress adaptation.In this thesis,the molecular network of flavonoids synthesis of cold-acclimated calli in Saussurea laniceps 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 S.laniceps callus,and reached peaks on 9th day,which was 1.7 times that of the control.This study identified 31 unigenes related to regulation of flavonoids pathway based on transcriptome data and DEG analysis,and discovered anthocyanins and isoflavones are two important branches of flavonoid biosynthesis in S.laniceps.The expressions of PAL,C4H,4CL,F3’H,IFR,OMT1,UGT,MRP3 and other genes were up-regulated after low temperature treatment,which was consistent with the increase of flavonoid synthesis under low temperature.2.Screening of reference genes.According to comprehensive analysis of △Cq method,geNorm,NormFinder and Bestkeeper based on transcriptome data,results showed that UEV1D and EF1B is two stable genes among the 13 candidate reference genes,while UBC36,ELF5A,and 7 are weak stability of genes in low temperature.Moreover,Actin7 was the most unstable expression gene,and the rest of 6 genes are intermediate stable expression genes in low temperature.3.Clone and genetic transformation of key genes.The CDS sequences of isoflavone reductase gene have been cloned from calli of S.laniceps using PCR technology,and named as SlIFR.Bioinformatics analysis showed that SlIFR gene sequence size is 942 bp,belong to SDR gene family,and encodes proteins with NADP and Nmrk-like conserved domains.Phylogenetic tree analysis showed that the SlIFR and Artemisia annua AaIFR proteins are highly homologous.Meanwhile,the prokaryotic expression of SlIFR protein was induced successfully,with a molecular weight of 52.6 kDa.The eukaryotic expression vector of pCAMBIA2300-SlIFR-GFP was successfully transformed into clonal tobacco by agrobacterium-mediated leaf disc method.This study preliminarily clarify that the metabolic pathway of flavonoids biosynthesis induced by low temperature in alpine plant Saussurea laniceps,providing a new genetic resources to breed new varieties of trees,ornamental plants,and medical plants through transgene technology. |
