Gene Expression And Single Nucleotide Polymorphisms Analysis Of Polyphenol Oxidase Genein Family Camellia Sinensis

Polyphenol oxidase(PPO)is a type of copper-containing oxidoreductase,which is the key enzyme in the formation of black tea quality during its processing.In this research,PPO gene were cloned and characterized in Camellia sinensis,and the expression conditions of PPO were optimized.The relative expression of PPO in different tea cultivars was analyzed.The single nucleotide polymorphisms of 94 Camellia sinensis cultivars were analyzed.The main results are as follows:1.The nucleic acid and amino acid sequences of PPOs genes are analyzed,and the physicochemical properties and structural functions of the PPO genes encoding protein are also predicted by bioinformatics method.The smallest relative molecular weight in the amino acid sequence transcribed from the PPO gene family is PPO2,which is 22.01 KD,and the largest is PPO1,which is 65.9 KD.The PPO1 and PPO3 encoded proteins are most likely to be localized in the chloroplast,and the two proteins are predicted to contain chloroplasts.The encoded proteins belonged to hydrophilic and unstable lipid binding proteins and had no signal peptides,which do not enter the endoplasmic reticulum and Golgi secretion pathway after synthesis,directly released into the cytoplasm.The proteins encoded by PPO2 and PPO4 are most likely to localize to other positions in non-chloroplasts and non-mitochondria.The proteins encoded by PPO1 and PPO3 genes may have one or more transmembrane helices.2.RNA is extracted from the leaves of Hongyafoshouand the Taoyuandaye Camellia sinensis as the material,and cDNA is reverse transcribed.The primers are designed by querying the base sequence of PPO gene through the published tea genomedatabase.A specific band of about 1800 bp is obtained by PCR technique.The DH5? competent cells are successfully transferred into the PPO recombinant plasmid,and then transfected with BamHI and Not I restriction enzymes.The expression vector Bl21 is expressed in fusion expression.The recombinant protein is induced by TPTG.The recombinant expression rate is 0.2mM at IPTG,the induction temperature is18?,the induction time is 6H,and the expression is the best.3.PPO enzyme activities in different tea cultivars differ geatly.Among the tested varieties,the PPO enzyme activities of Zhenghedabai,Hainandaye,Taoyuandaye,Ruchengbaimao and Xiuhong are more than 40 OD�g-1�min-1,and they are suitable for black teaprocessing;Pingyangtezao,Anjibaicha,Fuxuan 9,Jvhuachun,Yusun,Jinguanyin,Xianyuzao,Longjing43,Nanjiang 1,Hanlv,Yulv,Qiancha 8,Liubeixiang,Fuda 61 and Zhongcha 102 tea cultivarhave an enzyme activity of less than 5.0OD�g-1�min-1,andthey are suitable for green teaprocessing.Seasonal variation of PPO enzyme activities inZhuyeqi,Taoyuandaye and Bixiangzao tea cultivars are as follows:summer > autumn > spring.4.The relative expression of PPO1 gene ranged from 0.24?523.04,the highest expression variety is Zhenong 139,and the lowest is Meitan 5.The overall relative expression of PPO2 ranged from 0.24?160.02.The highest expression variety is Zhenong139 cultivar and the lowest value is Meitan 5 cultivar.PPO3 gene expression is higher in the samples of Taoyuandaye,Liubexiang,Hongyafoshou,Zijuan,Fudingdahao,Zhenong139 and Gaoqiaozao.The relative expression of PPO3 gene is 0.13?17451.00.The highest expression is Zhenong 139 and the lowest is Xiaoyefuding.The over all gene expression of PPO4 is low in all tea cultivars.It may be induced by other external factors.Correlation analysis showed that the relative expression of PPO1 gene is positively and significantly correlated with that of PPO2(r=0.770,p<0.01)and that of PPO3(r=0.809,p<0.01).The relative expression of PPO2 gene is also positively correlated that of PPO3(r=0.755,p<0.05).There is no significant correlation between PPO enzyme activities and the relative expression of PPO1-4 gene.5.A single nucleotide polymorphism(SNP)analysis of PPO gene is carried out in 94 different tea cultivars.The polyphenol oxidase gene has 1,800 bp and a total of 92 SNP sloci is found,1 SNP-site per 20 bases on average.It is indicated that PPO gene has a high level in genetic diversity.The transition type study is A?G,C?T,and the transversion type is A?C,A?T,G?C.There are 62 SNPs were synonymous mutations,and 30 SNPs are non synonymous mutations,which results in amino acid variation and accounts for 31.5%.By using SNP loci among different breeds that 94 varieties are successfuly identified.Taoyuandaye and Hainandaye cultivars with high enzyme activity are mutated at positions 955,678 and 1522.