Identification Of Polyphenols Compounds And Discovery Of Hydrolysable Tannin Metabolism Gene Cluster In R. Chingii

Rubus chingii Hu is a perennial woody plant of the genus Rubus in Rosaceae.The dried immature fruit is a kind of medicinal material into the kidney meridian.A total of 61 polyphenolic compounds,including 29 hydrolyzed tannins,15 flavonoids,11 phenolic acids and 6 other compounds,were isolated and identified from different tissues and organs by LC-MS in R.chingii.Among them,the accumulation of flavonoids and hydrolysable tannins(HTs)has caused unique material differences between various tissues.At the same time,a 231.21 Mb chromosomal-level genome of R.chingii was annotated by the third-generation sequencing technology,which provided an important genetic resource for understanding the metabolic pathway of HTs.The genomes of 16 representative species(including R.chingii)were selected for comparative genomic analysis,and a total of 4763 unique genes were identified in R.chingii,some of which were enriched by GO in the HTs pathway,such as "UDP-glucose: glucosyltransferase activity"(GO:0003980,P <0.05),"superoxide metabolic process"(GO:0006801,P <0.01),"methyltransferase activity"(GO:0008168,P <0.001)and " oxidation-reduction process,acting on CH-CH bond formation"(GO:0016627,P <0.001).According to the key gene information of the biosynthesis pathway of HTs,we further analyzed the gene families,and identified 139 UGT genes,56 SCPL genes,39 CXE genes and 57 PPO genes.Interestingly,a tandem gene cluster was found on chromosome 2,which may be closely related to HTs synthesis,including 11 UGT genes,6 SCPL genes and 8 CXE genes.According to the correlation analysis between gene expression level and hydrolytic tannin content,there is a high correlation between gene expression level and HTs content in this region.In order to further verify the function of this gene cluster,one CXE gene(LG02.4102)and one UGT gene(LG02.4273)were selected for in vitro enzyme activity analysis according to the function prediction and gene expression level of related gene evolutionary tree.The results of enzyme activity in vitro showed that LG02.4102 and LG02.4273 had the activity of hydrolyzing PGG and catalyzing the glycosylation of gallic acid,respectively.Comparative genomic analysis showed that this tandem gene cluster was conserved in the genomes of tannin-rich species such as strawberry and black raspberry,but expanded in the genomes of tannin-deficient species such as rose and apple to varying degrees,and even deletion of key genes such as tannase gene was found in apple.The results of correlation analysis,in vitro enzyme activity detection and comparative genomics analysis with Rosaceae showed that this cluster is the key region to regulate the biosynthesis of hydrolyzed tannins.