Transcription Regulation Of Polyphenol Oxidase Gene And Its Expression Analysis During The Withering Process Of Fresh Tea Leaves

Polyphenol oxidase(PPO)is an important terminal oxidase in tea plants(Camellia sinensis),which plays an important role in the catalytic oxidation of polyphenols in tea processing.However,the transcriptional regulation mechanism of PPO gene in tea plants is still unclear.In this paper,three tea varieties with different PPO activities were used as materials to screen the transcription factors that may regulate the expression of PPO gene in tea.Transcription factor Cs MYB12/48 were cloned and their function were verified.At the same time,the effects of different light quality treatments on the withering of fresh tea leaves were studied.The major results are showed as following:1.The transcriptome data of three tea varieties with different PPO activities were analyzed and compared.5153 differentially expressed genes in “TYDvs BXZ”were identified from the expression profiles.There are 3,986 differentially expressed genes in “TYDvs ZYQ”& there were 2059 differentially expressed genes in “ZYQvs BXZ”.Nine transcription factors with the same(or opposite)trend of PPO expression activity as those of three tea varieties were screened from these differential genes,which can be used for subsequent experimental analysis.2.Bioinformatics analysis shows that the total length of the nine transcription factors is 1080 bp for WRKY27,1446 bp for ARR12,and all others are below 1000 bp.Most of the start codons are ATG,the start codon of MYB5 transcription factor is GCT,and the stop codons are TAT,TAA and TGA.The protein encoded by the nine transcription factors are hydrophilic non-secretory proteins,and there are no signal peptide,so the protein function in cells.The prediction results of secondary and tertiary structures of protein show that transcription factors are mainly composed of α-helix and irregular curl,and only a small part of β-sheet structure exists,among which the protein secondary structures of MYB12 and MYB48 do not have β-sheet phenomenon.MYB5,MYB12 and MYB48 belong to MYB family.Because MYB5 start codon is not ATG,its protein amino acid translation function is worse than other transcription factors.WRKY27 belongs to Wrky family and b HLH51 belongs to b HLH family.3.The full length of Cs MYB12 is 444 bp,the encoded amino acid length is 147 AA,and the molecular weight is 16.95 k Da.The full length of Cs MYB48 is 750 bp,the encoded amino acid length is 249 AA,and the molecular weight is 28.93 k Da.Cs MYB12 is a basic protein,while Cs MYB48 is an acidic protein.They are unstable and contain a MYB domain unique to MYB transcription factor.Subcellular localization experiments showed that Cs MYB12 and Cs MYB48 were located in nuclei and belonged to nuclear proteins.Transcriptional activity experiments of tobacco and yeast showed that Cs MYB12 and Cs MYB48 had self-activation activity.The Cs PPO1 promoter was cloned,and the binding elements existed in the sequence were analyzed.It was found that Cs MYB12 and Cs MYB48 might activate the downstream gene by combining with the MYB binding elements on the promoter of Cs PPO1 by Dual-luciferase assay.4.The PPO activity of withered leaves under different light treatments is quite different,among which the PPO activity of withered leaves under yellow light treatment is higher than that of the control at 3,6 and 12 hours,and reaches the maximum at 3 hours.The q RT-PCR analysis showed that the PPOs gene expression of withering leaves under different light quality treatments was different,and the PPO1 and PPO3 gene expression of yellow light withering were higher than that of the control at 6h,and PPO4 gene expression was higher than that of the control at 12 h.