Physiological Indices And Gene Expression Of Flavonoid Metabolism At Different Development Stages In Lycium Chinense Fruits

As one of the secondary metabolites,plant flavonoids are the basic material for the color formation of flowers,fruits,seeds and other organs.During the growth and development of plants,flavonoid metabolism regulates the normal growth and development of plants and plays an important role in plant adversity.Lycium chinense fruits contain a variety of nutrients,such as polysaccharides,flavonoids and vitamin C.It is necessary to explore the metabolism of flavonoids in Lycium chinense fruits,which can provide a theoretical basis for the nutrition and quality of Lycium chinense fruits.In this study,the fruit development stage of Lycium chinense was divided into seven stages: early fruit,cyan fruit,green fruit,change color fruit,yellow fruit,red early fruit,and red ripe fruit.The physiological and molecular patterns of flavonoid metabolism in different developmental stages of Lycium chinense fruits were studied.The results were as follows:(1)The contents of flavonoids,catechins and kaempferol were the highest in Lycium chinense fruit.The contents of flavonoid metabolites were different in different development stages of Lycium chinense fruit.Flavonoid content was higher in cyan fruit stage and change color fruit,anthocyanin content was higher in red early fruit and yellow fruit,polyphenol content was higher in early fruit stage and change color fruit,catechin content was higher in yellow fruit and change color fruit,kaempferol content was higher in cyan fruit stage,red early fruit and red ripe fruit,isorhamnetin content was higher in yellow fruit,hesperidin content was higher in early fruit stage and cyan fruit stage,quercetin content was higher in REF(6).(2)The activities of flavonoid metabolizing enzymes showed an "S" trend during the development of Lycium chinense fruits.Among them,the activities of PAL,C4 H,F3H,F3'5'H and ans were the largest in red early fruit,the smallest in green fruit,the activities of CHS and CHI were the largest in red early fruit,the smallest in green fruit and change color fruit,the activities of 4CL,F3'H and FLS were the largest in early fruit stage,and the smallest in green fruit and change color fruit.(3)The biological information of early fruit stage,green fruit stage,change color fruit,yellow fruit and red ripe fruit in five developmental stages of Lycium chinense fruits were analyzed based on transcriptome sequence.A total of 175,810 Unigene were assembled effectively with an average length of 661.4 bp.116,591 unigenes were annotated in different databases,including 179 flavonoid metabolic pathways including anthocyanin,flavonol,flavonoid and phenylpropanoid synthesis,and 10 different genes of flavonoid metabolic pathways were screened out.(4)In the fruit development stage of Lycium chinense,the relative expression of4 CL,CHS,CHI,F3'H and FLS genes was the highest in cyan fruit,and the expression of PAL,F3 H and ANS genes was the highest in green fruit.Among yellow fruit,red early fruit and red ripe fruit,the expression of 8 genes(except C4 H and 4CL)was the lowest.(5)Two flavonoid metabolizing enzyme genes 4CL and CHI were cloned by RT-PCR.The full-length c DNA sequence of Lycium chinense 4CL is 1927 bp,including ORF 1716 bp(encoding 581 amino acid).The c DNA sequence of CHI gene of Lycium chinense is 695 bp,including an ORF 663bp(encoding 220 amino acids).The functional prediction of 4CL protein has Co A binding site and acyl activator consensus motif.CHI protein belongs to chalcone 3 family at 16-218 site.(6)The 4CL and CHI genes of Lycium chinense were connected to the prokaryotic expression vector.When 1mmol/L IPTG was added and the temperature was 37 ?,the expression of 4CL and CHI fusion protein was the highest.The results showed that both 4CL and CHI genes could induce the expression of protein in prokaryotic expression vector.