| Jujube,originated in China,is the first dried fruit and an important economic forest tree,and have a long history of cultivation and rich germplsm resources.Jujube fruit is rich in a variety of biological active compounds,and is a traditional “homology of medicine and food” nourishing food.Flavonoids are one of the important functional nutrients in jujube.In this study,semi-red fruits of 245 jujube germplasm resources of different types,fruits and organs at different developmental stages of ‘Hupingzao’ and‘Yeshengsuanzao’ were selected as test materials,Ultra-high performance liquid chromatography(UPLC)method was used to detect flavonoid compositions and contents in different germplasm.Based on metabolomics and transcriptome techniques,key metabolites and structural genes of flavonoid metabolic pathway were excavated,and the key genes were cloned,analyzed and functionally validated.Yeast two-hybrid technology was used to screen the interacting proteins.These studies can provide theoretical reference for further research on the metabolism and regulation mechnism of flavonoid in jujube fruit.The main results are as follows:1.UPLC method was used to systematically identify the flavonoid compositions and contents of different developmental stages fruits,different organs and 245 different types germplasm fruits,and antioxidant activity of 55 germplasm.The results showed that the total flavonoid content(TFC)and compositions of jujube fruit decreased with fruit development.A total of 13 flavonoid compositions were detected in all samples,including 12,11 and 10 in fruit,leaves and hanging stem,respectively.Flavonoid content in leaves was the highest,followed by hanging stem,and fruit was the lowest.Catechin,epicatechin and rutin were the main flavonoid compositions in fruis;quercetin and rutin were the main compositions in leaves;and catechin and rutin were the main compositions in hanging stem.In terms of different types,the flavonoid content in sour jujube was the highest,followed by intermediate type,and the cultivated jujube was the lowest.The flavonoids extracted from jujube fruits evaluated by FRAP,DPPH and ABTS methods had different antioxidant activities,and flavonoid content of different germplasm types were consistent with their antioxidant activities.By cluster analysis,245 germplasms were divided into 4groups with ultra-high,high,medium and low flavonoid content,and 17 excellent germplasms with hign flavonoid content were screened out.2.Taking different developmental stages fruits of Ziziphus jujuba cv.Hupingzao and Ziziphus spinosa cv.Taigusuanzao as test materials,flavonoid metabolites were analyzed based on widely targeted metabolomics.The results showed that 123 metabolites were identified,including 9 subclasses: 40 flavonols,37 flavones,12 anthocyanins,9 dihydroflavones,8 flavanols,7 flavone carboglycosides,5dihydroflavanols,3 isoflavones,and 2 chalcones.With fruit development,most of differential flavonoid metabolites(DFMs)were down-regulated;compared with ‘Hupingzao’,the DFMs of ‘Taigusuanzao’ were mostly up-regulated in the same period.KEGG pathway annotation and enrichment results showed that flavonoid biosynthesis pathway and secondary metabolite biosynthesis pathway were the main pathways of flavonoid synthesis in jujube;and the accumulation difference of flavanols was the main reason that made the flavonoid content in sour jujube be higher than that in Chinese jujube.3.Taking semi-red ripening stage fruits of ‘Hupingzao’ and ‘Taigusuanzao’ as test materials,the key genes of flavonoid metabolism were explored based on transcriptome technology.The results showed that transcriptome databases of ‘Hupingzao’ and ‘Taigusuanzao’ fruits were constrcted.The Clean data of each sample was more than 7.92 Gb,and the percentage of Q30 base was more than 94.68%.A total of 3476 differentially expressed genes(DEGs)were screened form the two groups of samples(H vs.S).Compared with ‘Taigusuanzao’,there were 1939 up-regulated genes nad 1737 down-regulated genes in ‘Hupingzao’.A total of 59 genes related to flavonoid metabolism were obtained by screening,among which 8 genes,CHS1,CHS2,CHI,F3 H,F3’H,DFR,ANS and UFGT,were up-regulated in ‘Taigusuanzao’.The expression levels of CHS,F3 H and DFR were high and the differences were large.Therefore,it is speculated that these genes play a key role in the synthesis of flavonoids in jujube.4.Taking semi-red ripening stage fruits of ‘Taigusuanzao’,c DNA from sour jujube fruit was used as template,we cloned the two genes of Zs CHS and Zs DFR.The expression characteristics and genetic transformation of Arabidopsis thaliana were analyzed to verify their function.The results showed that the CDS sequences of Zs CHS and Zs DFR were 1170 bp and 1026 bp,respectively.Multiple alignment of amino acid sequences and phylogenetic tree analysis showed that Zs CHS and Zs DFR were most closely related to Zj CHS and Zj DFR,respectively.In fruits of different germplasm,the expression levels of the two genes and flavonoid content were basically consistent.By comparing the expression levels of Zs CHS and Zs DFR in different organs,it is speculated that the expression levels of these two genes are tissue-specific.CHS may play a key role in falvonoid synthesis in fruits and kernels,and DFR may be a key enzyme in flavonoids synthesis of flowers and fruits.We successfully constrcted overexpression vectors of Zs CHS and Zs DFR and transformed them into Arabidopsis thaliana.Compared with wild-type arabidopsis thaliana,the phenotype of transgenic arabidopsis thaliana plants did not change signidicantly,but the content of flavonoids in leaves of all transgenic lines were significantly higher than that of wild-type arabidopsis thaliana,suggesting that the overexpression of these two genes could increase the content of flavonoids.5.The fruits at different developmental stages of ‘Hupingzao’ were selected as test materials,we successfully constructed c DNA yeast two-hybrid library of jujube fruit.The library capacity was 1.28 × 107,the recombinant rate was 100%,the inserted fragment was over 1000 bp,and the library titer was 5.0 × 107.The decoy vector PGBKT7-Zj CHS was successfully constructed,and it was found that the vector had no self-activation activity.By transforming yeast,we screened 29 positive clones,all of which interacted with Zj CHS protein.These proteins mainly include seven categories,such as hormone metabolism proteins,carbohydrate metabolism proteins,amino acid metabolism proteins,photosynthetic pathway proteins,protein metabolism,mitochondrial proteins and unknown proteins.These proteins may play a role in regulating growth and development,metabolism of active substances and stress.The mechanism of interaction between these proteins and Zj CHS,such as how the interaction affects flavonoid metabolism,needs further study.The library provides the basis for further study of protein interaction and facilitates the study of gene function. |
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