Mechanism Analysis Of ?-Glycosidase In The Degradation Of Anthocyanins From Brunfelsia Calycina And Arabidopsis Thaliana

Anthocyanins are the main pigments of horticultural crops,which can make photosynthesis of plants normal and protect from ultraviolet light,and have significant preventive and health functions for a series of human diseases.Therefore,Studies on anthocyanins have received extensive attention from researchers.At present,the species,chemical structure,coloration mechanism,biosynthetic pathway,cloning and expression of key enzyme genes,and the factors affecting the internal and external factors of anthocyanins have been studied in detail,but the degradation of anthocyanins research is very rare.Anthocyanin degradation plays an important role in the color regulation of plant flowers,leaves and fruits.It is of great theoretical and practical significance to investigate the degradation of anthocyanins and its regulation mechanism.In this paper,a glycosidase was isolated and purified from the Brunfelsia calycina,and it was determined to be ?-xylosidase by protein sequencing,and the gene sequence encoding the protein was cloned.The enzymatic properties of the enzyme and its role in the degradation of anthocyanin in Brunfelsia calycina were systematically analyzed.A large amount of Arabidopsis thaliana is used as a material to analyze key genes and metabolites of anthocyanin degradation based on transcriptome and metabolome,and to further analyze the role of related genes in the degradation of anthocyanins by deleting mutants.The main findings are as follows:(1)Extraction and purification of ?-xylosidase protein,gene cloning and enzymatic properties,and role in the degradation of anthocyanin in Brunfelsia calycina.A glycosidase was isolated and purified from the petals of Brunfelsia calycina in the fading stage,and the enzyme was identified as ?-xylosidase(EC3.2.1.37)by mass spectrometry.The enzymatic properties of the enzyme showed that the optimum temperature was 40 ?,the optimum p H was 5.0,and the optimal substrates were p NPGa and p NPX;the purified enzyme was reacted with the natural substrate,followed by HPLC and GC/MS techniques.It is elucidated that the enzyme can cleave the anthocyanin glycosidic bond,thereby releasing xylose and galactose monosaccharide,indicating that Bc Xyl is involved in the degradation process of anthocyanin,and cloning Bc Xyl according to the amino acid fragment using 3'5'race technology.Full-length,bioinformatic analysis showed a higher similarity to the glycosidase associated with the reported anthocyanin degradation.The results of q PCR showed that Bc Xyl and Bc Prx01 which can degrade anthocyanin were upregulated at the same time,and they were all localized in vacuoles.In addition,a certain degree of anthocyanin degradation was observed in the transient expression of Bc Xyl in the leaves of Brunfelsia calycina and red tobacco.(2)Based on transcriptome RNAseq sequencing to analyze the degradation of anthocyanin-related genes in Arabidopsis thaliana and the functional verification of corresponding mutants.The blue light treatment of Arabidopsis thaliana seedlings induced the synthesis of anthocyanins and the laccase and glycosidase genes were significantly up-regulated during the degradation of anthocyanins by RNAseq sequencing.The results of q PCR verification were basically consistent with those of transcriptome sequencing.The phenotypes of bxl1,bxl6,bglu5,bglu15,bglu17,bglu18 and bglu44 deletion mutants were observed.It was found that the decline rates of three mutants of xyl6,bglu18 and bglu44 in the degradation of anthocyanins were lower than those of wild-type Arabidopsis thaliana.These three genes are likely to play a role in the degradation of Arabidopsis thaliana anthocyanins.(3)Analysis of metabolites during anthocyanin degradation in Arabidopsis thaliana seedlings based on metabonomics sequencing.The blue light treatment of Arabidopsis thaliana seedlings induced the synthesis of anthocyanins,and the metabolites during the degradation of anthocyanins were analyzed by extensive targeted metabolome techniques.Cyanidins were found in anthocyanin metabolites.The compound may be an important component of Arabidopsis thaliana anthocyanins,among which Cyanidin-3-O-malonylhexoside,cyanidin-O-acetylhexoside(Cyanidin-Oacetylhexoside),cyanidin-O-hexosyl-O-hexosyl-O-hexoside and cyanidin-3-O-glucoside(The glycosidic bond of a compound such as Cyanidin-3-O-glucoside)is cleaved by a glycosidase to cause degradation of anthocyanin.Further combining RNAseq results to find genes closely related to key metabolites,it was found that the six genes Glycosyl hydrolase superfamily protein(AT4G16260),Peroxidase 52(AT5G05340),Peroxidase 54(AT5G06730),Peroxidase 15(AT2G18150)and Peroxidase 37(AT4G08770)may be closely related to the metabolism of cyanidin-3-O-malonylhexoside;Peroxidase 37(AT4G08770)may be closely related to the metabolism of Cyanidin-O-acetylhexoside.The mechanism of Arabidopsis thaliana anthocyanin degradation was further elucidated from the perspective of metabolite and transcriptome binding.