Cloning And Function Analysis Of CYP82Ds Genes From Camellia Sinensis

Purine alkaloids(PAs)are important secondary metabolites of tea plant,mainly including caffeine,theobromine,theophylline and theacrine,which have significant effects on stress resistances of tea plant and quality of tea products.The pathway of caffeine biosynthesis and its related genes have been clarified in tea plant,but the catabolic pathway and key genes are still unclear.In this study,cytochrome P450 82Ds(CYP82Ds),the candidate genes related to caffeine catabolism were obtained from leaves with different maturity harvested from ’Fuding Dabaicha’,’Lingyun Baihao’ and Camellia kucha through PAs detection,transcriptome sequencing and weighted gene co-expression network analysis(WGCNA).The main results are as follows:(1)Caffeine and theacrine were the dominant alkaloids in leaves of ’Fuding Dabaicha’and C.Kucha,respectively,and their contents decreased with an increase of leaf maturity.The dominant alkaloids of ’Lingyun Baihao’ varied with leaf maturity,i.e.,caffeine was dominant in young leaves while theacrine did in mature leaves,and an obvious alterantion occurred during leaf maturity.6839 differentially expressed genes(DEGs)were screened from the various maturity leaves harvested from three tea plant resuorces,and 19 modules with different expression patterns were obtained through WGCNA.Among them,pink and lightgreen modules were significantly related to content of caffeine and theacrine,respectively.DEGs in pink module were mainly related to ’porphyrin and chlorophyllin metabolism’,’synthesis of secondary metabolites’ and ’purine metabolism’ pathways;DEGs in lightgreen module were enriched in ’m RNA regulatory pathway’,’pyruvate metabolism’ and ’carbon fixation of photosynthetic organisms’.The candidate hub genes in pink and lightgreen modules included epoxide hydrolase B(EPHB),metallothionein-like protein 1(MT1)and serine/threonine-protein kinase 3(RKF3),as well as CYP82 D,glyceraldehyde-3-phosphate dehydrogenase(G3PB)and phospholipase D zeta 2(PLDZ2),respectively.(2)CYP82D1,CYP82D2 and CYP82D3 genes were cloned from leaves of ’Fuding Dabaicha’,’Lingyun Baihao’ and C.kucha.Their open reading frames were 1572 bp,1560bp and 1599 bp,respectively,encoding 523,519 and 532 amino acid residues.Two transmembrane helices were predicted in CYP82D1 and CYP82D2,while only one in CYP82D3.Amino acid sequences of CYP82D1/3 were similar in different tea plant resources,but 50 amino acids of CYP82D2 in ’Fuding Dabaicha’ were found different in comparion with ’Lingyun Baihao’ and C.kucha.q PCR results showed that the expression of CYP82D1 in ’Fuding Dabaicha’ and ’Lingyun Baihao’ was up-regulated,while that in C.kucha was down-regulated.Expression of CYP82D3 was detected in all sampled leaves of ’Lingyun Baihao’ and the significantly highest level in old leaves,but not witnessed in leaves of C.kucha.CYP82D3 expressed similarly in all leaves of’Fuding Dabaicha’.The expression levels of CYP82D2 in ’Lingyun Baihao’ and C.kucha were significantly higher than those in ’Fuding Dabaicha’,and highest levels were detected in old leaves of ’Lingyun Baihao’ and mature leaves of C.kucha,which were45760 and 101446 times higher than those in tender leaves of ’Fuding Dabaicha’,respectively.The expression trend of CYP82D2 was consistent with that of theacrine content.The element analysis of promoter showed that there were a large number of elements related to light response,environmental stress response,hormone response,MYB and other transcription factor binding sites in the promoter region of CYP82D2 in three tea plant resources,in addition to the essential basic core elements of higher plant promoters.The number of MYB-like transcription factor binding sites in CYP82D2 promoters of ’Lingyun Baihao’ and C.kucha was more than that in ’Fuding Dabaicha’,and low temperature responsive element LTR only existed in ’Lingyun Baihao’.The differential expression of CYP82D2 in leaves of various tea plant resources might be related to the difference of promoter elements.(3)The CYP82D2(CYP)and a cytochromes P450 reductase(CPR)obtained from’Lingyun Baihao’ were inserted into the yeast co-expression vector p AO815 and was designated as p AO815-αCYP-αCPR.The obtained vector was transferred into Pichia pastoris GS115 strain.After being induced by 1% methanol,the two target proteins were achieved,but their concentrations were too low to support the enzyme activity analysis.The expression vectors of p Cold-TF-CYP and p Cold-TF-CPR were constructed and transformed into E.coli Origami B(DE3),respectively.After IPTG induction,the two target proteins could be expressed properl in E.coli.The CYP82D2 was optimally induced with 0.4mm IPTG at 15°C for 24 h,and the CPR was optimally induced with 0.6mm IPTG at 15°C for 24 h.Purine alkaloids were added into the mixed bacterial solution containing vector p Cold-TF-CYP and p Cold-TF-CPR,but no product was detected.In order to verify the enzyme activity of the CYP82D2,the in vitro reaction system should be further adjusted.In addition,subcellular localization test showed that CYP82D2 was located in endoplasmic reticulum.(4)To study the effect of CYP82D2 on caffeine catabolic pathway,antisense oligonucleotide(as ODN)technology was used to knock down the expression of CYP82D2 in tea leaves of ’Lingyun Baihao’.It was found that the content of caffeine and theobromine treated with CYP82D2-as ODN was higher when the expression of CYP82D2 was inhibited,indicating that CYP82D2 participated in the caffeine catabolism process in ’Lingyun Baihao’.