Cloning And Analysis Of Pyridoxal Kinase And Pyridoxine 5'-phosplhate Oxidase Gene From Tobacco

Vitamin B6?VB6?was a generic term for pyridine compounds that could be converted to each other.It included Pyridoxine?PN?,Pyridoxamin?PM?,Pyridoxal?PL?and their corresponding phosphate forms Pyridoxine-5'-phosphate?PNP?,Pyridoxamine-5'-phosphate?PMP?and Pyridoal-5'-phosphate?PLP?.Among the six forms,PLP was a coenzyme of more than 140 enzymes in the cell.Animals themselves could not synthesize VB6,which required to get VB6 from food and synthesized PLP through the metabolic transformation pathway.There were two ways to synthesize VB6 in nature,one is the DXP-dependent path and the other is the DXP-independent approach.Plants synthesized PLP by DXP-independent pathways.PLP was metabolized into different forms of VB6 through a series of enzymes in plants.In this process,VB6 phosphatase,PL reductase,PM-pyruvate transaminase,pyridoxal kinase?PLK,Pyridoxal kinase?and pyridoxine phosphate?PNPO,Pyridoxine 5'-phosphate oxidase?played a vital role.PLK could phosphorylate PN,PM and PL to PNP,PMP and PLP.PNPO could oxidize PNP and PMP to PLP.Plants were the main food source for animals and were the main source of VB6.The study of the metabolic transformation pathway of VB6 in plants was of great significance to the supply of human vitamin VB6.It was also necessary to study the key metabolic transformation enzymes of PLK and PNPO.PLK and PNPO had been cloned and identified in plant Arabidopsis thaliana to study the effect of other VB6 anabolic gene on the gene expression level by T-DNA insertion mutations,but they also lacked the effect on different species and different methods verification.In this study,PLK and PNPO studies were carried out using plant tobacco as a material,which could be used as a reference for the synthesis and metabolic transformation of vegetative VB6 in plants.In this study,the cDNA sequences of NtPLK and NtPNPO were predicted by using the PLK and PNPO cDNA sequences of Arabidopsis thaliana in tobacco ESTs.The accession numbers of NtPLK and NtPNPO were XM₀09804516.1 and XM₀09801441.1 respectively.The sequencing of NtPLK and NtPNPO were cloned to obtain the DNA fragment of the expected size.The sequencing results showed that the coding length of NtPLK was 1020 bp,and it encoded 339 amino acids,and its molecular weight was 37.4kDa,isoelectric point was 6.96.Amino acid sequence alignment found that relatived to mammals,insects and bacteria there was an extension of the N-side,enzyme activity sites at the amino acid residues was conservative.The gene spaned 1.2 kb DNA on the chromosome,containing 13 exons and 12 introns.The coding length of NtPNPO was 1620 bp,it encoded 539 amino acids,its molecular weight was 60 kDa and the isoelectric point was 8.03.Compared to mammals,insects,and bacteria,amino acid sequence alignment revealed a significant extension of about 280 amino acid residues at the N-terminus,which was the unclear YjeF_N domain.The gene spans 0.7 kb DNA on the chromosome and contained 14 exons and 13 introns.The expression level of NtPLK in tobacco leaves was the highest,and the expression level in root and stem was low and similar.NtPNPO had the highest expression level in tobacco leaves,lower in stem and the lowest in root.The target coding sequence of NtPLK and NtPNPO were inserted into pET32a?+?expression vector to express the expected protein in Rosetta E.coli.The crude enzyme solution of NtPLK showed an activity of pyridoxal kinase of 29.7 nmolPLP/mg/min with the substrate PL.The activity of the crude enzyme solution of NtPNPO showed that the activity of pyridoxal phosphate oxidase was 21.4 nmolPLP/mg/min with the substrate PMP.Enzyme activity confirmed that the clones encoded by our sequences were tobacco PLK and PNPO.The specific sequence of 490 bp in NtPLK and the specific sequence of 497 bp in NtPNPO coding band were selected,and their positive and negative sequences were inserted into the ends of pHANNIBAL's introns respectively.The RNAi vector was constructed.The best time for NtPLK RNAi to down-regulate the expression of NtPLK transcription level in tobacco leaves was 72 h by qRT-PCR.The best time for NtPNPO RNAi to down-regulate the expression of NtPNPO transcription level in tobacco leaves was 48 h.The expression levels of NtPLK,NtPNPO and NtPDX1 in tobacco leaves decreased by 50.9%,51.1% and 26.3% respectively,while that of NtPLR increased by 27.7%;NtPNPO,NtPNP,NtPLR and NtPDX1 transcription levels decreased by 68%,22.1%,29.5% and 33.9%,respectively after NtPLK RNAi treatment for 48 h.It was confirmed that the constructed RNAi vector successfully down-regulated the expression of the target gene.