Cloning And Analysis Of Vitamin B₆ Metabolizing Enzyme Pyridoxal Phosphate Phosphatase Gene In Tobacco

Vitamin B₆ comprises six interconvertible pyridine compounds,including pyridoxine?PN?,pyridoxal?PL?,pyridoxamine?PM?,and their phosphorylated forms:pyridoxine5'-phosphate?PNP?,pyridoxal 5?-phosphate?PLP?and pyridoxamine 5?-phosphate?PMP?.PLP is the active form of VB6 and it is the coenzyme of over 140 enzymes,participated in the catalytic response to amino acid as a coenzyme in many reaxtions.PLP is the catalytically active form of vitamin B₆,which plays an important role in maintaining biochemical homeostasis,including the metabolism of amino acids in organisms and the characteristics of its own chemical structure.The characteristics of active aldehyde group make it easy to combine with compounds containing amino,generating aldehyde imine class materials,which impact on normal life activities.Therefore,other forms in the VB₆ family must be converted to PLP.The enzymes that are normally involved in metabolism are ATP-dependent PL kinase and FMN-dependent PNP oxidase.The hydrolysis of PLP is an important mechanism to control the level of free PLP in cells,so the phosphatase that can catalyze the hydrolysis of PLP is of great significance in maintaining the concentration of PLP in the organism within a normal range.In this experiment,a model plant tobacco was used as the material,and the gene sequence of human-specific PLP phosphatase that had been reported and verified was used to find a tobacco PLP phosphatase gene?named NtPLPP?which was predicted by the blast on NCBI.The gene was 951bp in length and encoded 316 amino acids.The molecular weight of encoded protein was 34.157 kDa and the isoelectric point was 5.11.The functional domain prediction of its amino acid sequence showed that tobacco NtPLPP gene and human PLP phosphatase belonged to the same family,namely the HAD protein superfamily.Through the comparison of the homology of NtPLPP and multi-species amino acids,it was found to be different from other common phosphatases,However,It was consistent with the key conserved sites of PLP phosphatases of various species.In this study,pMal-c2x-NtPLPP prokaryotic expression vector was constructed and transformed into E.coli for prokaryotic expression.The target protein was successfully obtained in the supernatant and the target protein was analyzed for partial enzymology.The results showed that NtPLPP could catalyze the PLP to generate PL,and the optimum pH of the reaction when PLP was used as substrate was 7.5,the optimum temperature was 60?,and the enzymatic reaction time was 20 min.Using malachite green method to determine the enzyme activity of different substrate,it was found that the enzyme had a certain catalytic activity on these substrate,but it was the most active when PLP was the substrate.The specific sequence of 273bp in NtPLPP coding band was selected,and its positive and negative sequences were inserted into the ends of pHANNIBAL's introns respectively.The RNAi vector was constructed.The best time for NtPLPP RNAi to down-regulate the expression of NtPLPP transcription level in tobacco leaves was the time after qRT-PCR for 48h,and the expression level at transcriptional level decreased by 59%.The expression levels of NtPLK,NtPNPO and NtPLR in tobacco leaves decreased by 46%,38%and 49%respectively.Through the construction of NtPLPP-GFP expression vector of green fluorescent fusion protein,tobacco leaves were transiently infected with Agrobacterium transformation method,and the distribution of NtPLPP-GFP fusion protein in tobacco leaves was observed using LSM880 laser confocal microscope.It was found that in tobacco leaves,most of the PLP phosphatases were located in the chloroplast,and some other PLP phosphatases were located in the cell membrane and nucleus.