Cloning Of Polyphenol Oxidase From Tea Plant(Camellia Sinensis)and Its Response To Stress

Tea plant(Camellia sinensis)is an important economic crop in China.During the growth and development of tea plant,besides the low temperature stress,also facing the damage of tea pests,which seriously affect the yield and quality of tea in China.Therefore,it is of great significance for tea molecular breeding technology of insect resistance and development of the green pesticides to excavate the important defensive genes and analyze their mechanism of defense in tea plant.Polyphenol oxidase(PPO)is an important defense enzyme in plants and plays an important role in plant insect resistance.In this study,we cloned full length sequences of two PPO genes and two LAC genes by RT-PCR and RACE technology.The bioinformatics analysis method was carried out to analyze the sequences and qRT-PCR was used to analyze the expressions of these genes in different tissues of tea tree and different treatments.Preliminarily clarifying the expression patterns of those genes and their response to different treatments so as to provide a better candidate gene and theoretical basis for the future research of tea tree resistance gene engineering.The main results were as follows:1.Two full-length cDNA sequences of PPO genes were cloned from C.sinensis cv.Longjing 43 using RACE-PCR and RT-PCR based on the previous transcriptome sequencing,named CsPPO1 and CsPP02 respectively.Bioinformatics analysis suggested CsPPO1 and CsPP02 contain 1740 bp and 1788 bp open reading frame(ORF),encoding 579 and 595 amino acids respectively.The deduced CsPPO1 and CsPP02 proteins had the molecular weights of 64.6 kDa and 65.9 kDa and theoretical isoelectric points of 6.74 and 6.61,respectively.PPO gene family was conservative and had high similarity in different plants.CsPPO1 and CsPP02 both contain three conserved domains(tyrosinase,PPO1-DWL and PPO-KFDV).Phylogenetic analysis suggested CsPPOs have close genetic relationship with lotus flower and pokeweed,have distant genetic relationship with tomatoes and tobacco.2.The results of qRT-PCR suggested CsPPO1 and CsPPO2 have different expression patterns in tea plant tissues.CsPPO1 has the highest expression level in stem,CsPP02 has the highest expression level in root and lowest expression level in stem.The expression levels of CsPPO1 and CsPPO2 under different treatments also had difference.Although wounding and the infestation of Ectropis oblique(EO)treatments both induced the expression levels of CsPPO1 and CsPP02.JA treatment only induced the expression levels of CsPP02 and CsPPO1 had no response.The results of regurgitant treatment indicated that the regurgitant of EO significantly up-regulated the expression of CsPP02 compared to plants treated with wounding plus deionized water at 12 hr and 24 hr after the start of treatment,approximately 2.22 and 3.73 times,respectively.But,the expression of CsPPO1 did not differ significantly.Those results suggest CsPPO1 and CsPPO2 play important roles in tea plant defense,and the response of CsPPO2 under treatments is stronger,which indicates CsPPO2 may play more defence function in tea plant.3.In order to further excavate the polyphenol oxidase genes related to insect resistance in tea plant,two laccase genes were cloned by RT-PCR from tea plant cultivar 'Longjing 43' based on the transcriptome database of tea plants,named CsLAC4 and CsLAC12,contain 1665 bp,1743 bp and encode 555 and 581 amino acids respectively.Bioinformatics analysis showed that the molecular weights of CsLAC4 and CsLAC12 were 61.1 kDa and 63.9 kDa and theoretical isoelectric points were 8.34 and 9.18,respectively.The protein sequence of CsLAC4 and CsLAC12 all contain three typical copper binding domains and have signal peptides in the the N-terminal and belong to secretory protein.The phylogenetic tree indicated CsLAC4 and CsLAC12 were distributed to different evolutionary branching.QRT-PCR analysis showed that the expression patterns of CsLAC4 and CsLAC12 were similar in the different tissues of tea plant,both have the highest expression level in the stem.CsLAC4 has the lowest expression level in the flower and seed and CsLAC12 has the lowest expression level in the seed.JA,mechanical wounding and EO treatments all significantly induced the expression level of CsLAC4.However,JA and mechanical wounding treatments significantly suppressed the expression level of CsLAC12 and only EO treatment significantly induced the expression level of CsLAC12.These results suggested that CsLAC4 and CsLAC12 were involved in the tea plants respond to the exogenous hormone,mechanical wounding and the infestation of EO,but play different functions.The difference of expression level of CsLAC12 that significantly suppressed under mechanical wounding treatment and significantly induced under EO treatment indicating tea plants can specificity recognize the infestation of EO and CsLAC12may have a specific response to the oral secretion of the EO.