Cloning And Expression Of Vitamin B₆ Metabolizing Enzyme Genes PLK,PNPO And PLR In Camellia Sinensis

Vitamin B₆?VB₆?is a generic term for a group of 2-methyl-3-hydroxypyridines,which including Pyridoxine?PN?,Pyridoxal?PL?,Pyridoxime?PM?and their corresponding phosphate forms:Pyridoxine-5'-phosphate?PNP?,Pyridoxal-5'-phosphate?PLP?and pyridoxamine-5'-phosphate?PMP?.PLP is the most important activity form in VB₆,it is a coenzyme of more than 140 cellular enzymes involved in the synthesis and metabolism of various substances in organism.Plants can synthesize PLP through a DXP-independent pathway,at the same time PLP is metabolically converted by different forms of VB₆ in plants through a series of enzymes.In the processes,VB₆ phosphatase,PL reductase?PLR?,PL kinase?PLK?and PNP oxidase?PNPO?play a crucial role.At present,PLK,PNPO and PLR had been successfully cloned and identified in Arabidopsis thaliana.Through the T-DNA insertion mutation,the expression of VB₆enzyme genes was explored at gene level,these genes were explored under the low temperature,high light,drought,application of exogenous hormones and the other stress.The VB₆ research materials are mainly herbaceous plants such as arabidopsis,tobacco and the others.In this experiment,the PLK,PNPO and PLR studies were conducted by using tea as a material.It was an reference for the synthesis and metabolism of nutrient VB₆ in plants.In the study,the coding sequences?CDS?of arabidopsis thaliana PLK,PNPO and PLR were aligned in tea tree CDS library to find the predicted sequences of CsPLK,CsPNPO and CsPLR,and then the coding frames of CsPLK,CsPNPO and CsPLR were cloned.The result showed that CsPLK encoded a protein contains 344 amino acid residues,with a theoretical molecular weight of 37.9kDa and a theoretical isoelectric point of 6.51;CsPNPO encoded a protein contains 501 amino acid residues,with a theoretical molecular weight of 48.5kDa and a theoretical isoelectric point of 5.82.CsPLR encoded a protein contains 324 amino acid residues,with a theoretical molecular weight of 36.1 kDa and a theoretical isoelectric point of 9.10.The expressions of CsPLK in tissues of tea tree were as follows:leaf>stem=root;the expression of CsPNPO was leaf>stem>root.CsPLR is also leaf>stem>rootThe cloned CsPLK,CsPNPO and CsPLR coding sequences were imported into the pET22b expression vector and expressed in Escherichia coli Rosetta.Compared to the substrate PL,the enzymatic activity of pro-CsPLK was 4.853?mol PLP/mg/min;which was 9.17 times of the control group pro-22b with an enzyme activity of 0.529?mol PLP/mg/min.Compared to the substrate PMP,the pro-CsPNPO enzyme activity 4.375?mol PLP/mg/min was 12.72 times of the control group pro-22b with an enzyme activity of 0.344?mol PLP/mg/min.Enzymatic activity experiment confirmed that the proteins encoded by the sequences that we cloned were CsPLK and CsPNPO.Low temperature and drought stress were set for Longjing No.43.With the Quantitative Real-time PCR?QRT-PCR?we found that under the low temperature treatment the expression of CsPLK was increased first,then decreased gradually after 8 hours;the expression of CsPNPO was gradually decreased to the lowest level at 24 h,then recovered;the expression of CsPLR was also decreased and reached the lowest at 24 hour,but it was not significantly decreased in the early period.Under drought stress,the expression of CsPLK was decreased to the lowest on the 12th day,and recovered on the 6th day after rewatering;CsPNPO was down-regulated and reached the lowest point on the 12th day,then increased after rewatering;the expression of CsPLR was increased and reached the highest on the 12th day,and then down-regulated after 6 days of rewatering.It was confirmed that CsPLK,CsPNPO and CsPLR were involved in the adverse physiology of tea tree under low temperature and drought stress.