Omics Comparison Of Differential Catechin Metabolism In Tea Plants And The Molecular Mechanism Regulated By ANR Gene

In this study,the differential catechin metabolism of Camellia ptilophylla(MY)and Yingghong 9(YH)were utilized as materials.The differences in metabolite composition and corresponding enzyme genes expression between the two materials were revealed based on metabonomics and transcriptomics.The flavonoid contents and the expression of related key synthase genes were further determined by HPLC and quantitative PCR.On this basis,the key enzyme gene ANR,which was involved in the catechin synthesis pathway and differentially expressed between the two selected tea plants,was selected and cloned.The difference of catalytic function of prokaryotic expression enzyme was determined and compared,meanwhile,the specific antibody of ANR gene was developed and used for analysis the difference in translation level between the two materials by Western-blot technique.The characteristics of protein interaction between the cloned genes were determined via yeast two-hybrid technique as well as subcellular localization by transient expression system,respectively.In addition,with the help of transgenic technology,the cloned ANR gene was over-expressed in model plant tobacco and suppressed or knockout in tea callus via CRISPR-Cas9 transgenic techniques to elucidate the effect of ANR gene,the main results are as follows:(1)Metabolomics analysis and physicochemical validation.The analysis of targeted metabolomics showed that MY and YH had obvious different metabolic characteristics,and the differential metabolites mainly enriched in caffeine,phenylalanine,flavonoid and flavonoid,arginine and proline,anthocyanin and other secondary metabolic pathways.A total of 225 differential metabolites were obtained,of which 57 differential metabolites(33 down-regulated and 24 up-regulated)were involved in the metabolic pathways of phenylalanine and flavonoids,which including catechins and its derivatives,flavonoids,flavanones,flavonoids,and anthocyanins.According to the results of metabolomics,flavonoid was selected and further verified by HPLC.The results showed that catechin in MY was lower than that in YH,and reached106.02 mg/g and 181.51 mg/g,respectively.In terms of catechin composition,non-phenotypic catechins account for 70.48 % of the total catechin content in MY,especially the content of GCG reached 47.14 % of total catechin in.Catechin composition in YH is completely different from that in MY.The phenotypic catechin accounted for90.63 % of the total catechin in YH,the compound ECG exhibiting the highest content and accounted for 34.57 % of the total catechin.In addition,the contents of anthocyanidin and anthocyanin in MY were higher than those in YH,the former was 0.42 mg/g and 2.84 mg/g,the latter was 0.18 mg/g and 2.24 mg/g,and reached 2.33 and 1.27-fold higher than that of YH,respectively.(2)Transcriptome analysis and q RT-PCR verificationThe high throughput sequencing and parameter analysis of the transcriptome data indicated that the great differences existed in gene transcription level between MY and YH,and a total of 9212 differentially expressed genes were identified(4425 up-regulated and4787 down-regulated).KEGG annotation showed that 44 differentially expressed genes(5up-regulated and 39 down-regulated)were involved in flavonoid metabolism pathway.Among the key enzyme genes of flavonoid metabolism,the relative expression of PAL,4CL,CHS,F3 H F3'H,F3'5'H,FLS,DFR,LAR,ANS,ANR,UFGT in MY were lower than that of YH except for the up-or down-regulation of different members of UFGT family genes.According to the results of transcriptomic,12 candidate genes related to the metabolism of flavonoids were selected for q RT-PCR verification.The results indicated that the expression level of 4CL,CHS,F3'H,F3'5'H,FLS,DFR,LAR,ANS,ANR1,ANR2,UFGT2 and UFGT3 genes in MY were significantly lower than that in YH.In contrast,the expression levels of PAL,F3 H,and UFGT1 were significantly higher than that in YH.The comparative analysis showed that the genes expression determined by q RT-PCR were consistent with those observed in the RNA-Seq except for PAL,F3 H gene.(3)Cloning and enzyme activity determination of ANR geneTwo ANR family genes including ANR1 and ANR2 were cloned from MY and YHrespectively by RT-PCR.The open reading frame(ORF)of genes were 1014 bp and 1047 bp,and they showed more than 92% homology compared with the genes in the international database.The homology of ANR1 and ANR2 gene between the two materials was 99.6% and 98.6%,respectively,while the sequence homology between ANR1 and ANR2 in the same material was about 82%.The results of prokaryotic expression indicated that the ANR1 and ANR2 proteins were soluble proteins,and the corresponding products EC and GC were detected in the enzyme reaction system with delphinidin as substrate,and EGC and EC were detected with cyanidin as substrate.In addition,ANR1 fusion protein also produced GC when cyanidin used as substrate.(4)Protein expression,interaction and subcellular localization analysis of ANR geneThe corresponding antibodies of ANR1 and ANR2 were prepared in New Zealand rabbits using synthetic polypeptide according to the cloned gene sequence.The titers of the antibody were more than 1:80 000,and verified owned specified banding properties with the target protein.Western-blot results showed that the expression of ANR1 protein in MY was significantly higher than that in YH,but ANR2 protein in MY was slightly lower than that in YH with a not significant difference.Meanwhile,ANR1 and ANR2 protein showed no interaction analyzed by yeast two-hybrid system.Subcellular localization results showed ANR1 and ANR2 proteins were located in chloroplast,cytoplasm,cell membrane,and nucleus in Tobacco plant.(5)Transgenic functional verification of ANR geneThe over-expression of ANR1 and ANR2 genes in tobacco could significantly increase the contents of flavonoid in leaves and affect the flower color by adjusting anthocyanin content.Meanwhile,the expression of CHI,CHS,F3'H,DFR,LAR,ANS gene was found increased in transgenic tobacco leaves,while the expression of UFGT genes was decreased.The inhibition gene expression of ANR was verified by using CRISPR-Cas9 technique on tea callus.Sequence analysis of the target genome in callus showed that the directed mutation was successful.The gene expressing of ANR1 in resistant transgenic callus was decreased to 0.03,which is 35.25-fold lower than that of non-transgenic tissues.In addition,the expression of FLS,F3 H,F3'H,CHS,DFR,LAR genes also detected significantly decreased,while the gene expression level of PAL,4CL,UFGT were obviously increased.