Analysis And Expression Of CsPPT Genes And Their Effect On Tea Quality Formation

More than 3,000 years about tea plant(Camellia sinensis)cultivation made the tea germplasm resources extremely rich in China.For the different ingredients contained in fresh leaves,they could be made to different dry tea.The polyphenols,amino acids and caffeine were related to the quality of tea.Usually the ratio of polyphenols and amino acids,as an important indicator,is used to evaluate the quality of tea.In the synthesis pathway of polyphenols and amino acids in tea plant,phosphoenolpyruvate(PEP)acts an important role.As a substrate,PEP can be used to synthesis secondary metabolites such as catechins after they were transported into chloroplast,or can be used to synthesis of theanine when they kept into the cytosol by TCA cycle and ALA decarboxylation.Which made PPT(phosphoenolpyruvate/phosphate transporter)were important in the tea plants.In this study,CsPPT2 was cloned and compared with another transporter gene CsPPTl in tea plant.Also the expression of these two genes in different tissues,different growth stages,different tea varieties and different development period were compared.The results are as follows:(1)A full length cDNA sequence of phosphoenolpyruvate transporter gene(CsPPT2)was obtained from tea plant(Camellia sinensis)cultivar 'Baiye 1' by polymerase chain reaction(PCR)and rapid amplification of cDNA ends PCR(RACE-PCR).The length of nucleotide sequence of this gene was 1469 bp,containing a complete open reading frame(1218 bp)to encode 406 amino acids.Bioinformatic analysis showed that the molecular weight of the predicted protein was 44.6 kD,and the theoretic isoelectric point was 9.90.CsPPT2 had 6 trans-membrane regions,which may belong to the hydrophobic chloroplast trans-membrane protein.(2)After checked the sequence of the amino acids in 'Baiye 1','Longjing 43','Huangjinya' and 'Yingshuang',there is no mutant point in CsPPT1 and CsPPT2 genes.Reading from the poylogenetic tree of CsPPT result,CsPPT1 and CsPPT2 were divided into two subgroups in the phosphoenolpyruvate transporter family.The expression of CsPPT1 and CsPPT2 were compared in different stages and in different tissue.It showed that CsPPT2 was expressed in all tissues,including roots,leaves,stem and vein.The expression level in roots and mature leaves was higher than that in CsPPT1.But the expression level of CysPPT1 in young shoots was higher than that in CsPPT2.CsPPT2 was temporarily elevated at the beginning of the albino,but inhibited during the albino development,and CsPPT1 kept decrease in the whole albino stages.(3)The content of polyphenols,catechins,free amino acids was detected in four tea plant cultivation such as 'Baiye 1','Longjing 43',Huangjinya' and 'Qiannianxue'.The realative expression was checked during three stages as buds,a bud with one leaf and a bud with two leaves.After the analysis,it showed that the expression of CsPPT1 in buds were highest during the different stages in all four cultivars,but the CsPPT2 were opposite.It means that the function of CsPPT1 was replaced by CsPPT2 during the leaf development.The expression of CsPPT1 was much higher in 'Longjing 43 ' than that of the other three cultivars in all leaf development stages.Its expression in buds was 80-,200-and 40-fold higher than 'Huangjinya','Qiannianxue' and 'Baiye 1',respectively.In the stage of a bud with one leaf and a bud with two leaves its expression were 8-,6-,27-fold and 11-,4-,19-fold higher than other three cultivars.So the amount of CsPPTl may be related to the content of polyphenols and amino acids in tea leaves.The expression of CsPPT2 should be increased with the mature of leaves in 'Baiye 1',but it has been strongly inhibited during the albinism process.It means less PEP can be transported in the chloroplast,which brought the lower ratio of polyphenols and amino acids in 'Baiyel',and made the Baiyel' be a special cultivar.