Identification,RNAi And Subcellular Localization Of NtPLR Genes In Tobacco

Vitamin B6(VB6)is the general name of a class of interchangeable pyridine compounds,of which PLP(pyridoxal phosphate)mainly plays a coenzyme role and participates in the metabolism and regulation of more than 140 enzymes.As the main source of human intake of VB6,it is of great significance to study the metabolism and transformation of VB6 in plants.In plants,in addition to using the "DXP independent pathway" to de novo synthetic PLP,there is also a salvage pathway of PLP,which is achieved by a series of enzymes.Mutual conversion of VB6 and remedial synthesis of PLP,as well as maintaining stability of the VB6 state.The salvage synthesis of PLP is mainly involved three kinds of enzymes:ATP-dependent PL kinase(PLK),FMN-dependent PNP oxidase(PNPO),and NADPH-dependent PL reductase(PLR).PL produces PN under the action of PL reductase,and PN is the main component of vitamin B6 content in most vegetable-derived foods.Therefore,studying PL reductase is of great significance for clarifying the source of PN in plants.Moreover,PL kinase and PNP oxidase have been studied in detail in plants,and there are few studies on PL reductase in plants.Therefore,this study laid the foundation for a comprehensive understanding and the mechanism of exploring the metabolic transformation of the VB6 salvage synthesis pathway in plants.In this study,PLR sequences in tobacco were predicted by BLAST alignment using known arabidopsis PLR sequences.The sequence is composed of 1110 bp bases and encodes 369 amino acids.The molecular weight of the encoded protein is 41.1 KD and the isoelectric point is 9.59,belonging to the aldehyde ketone reductase superfamily.The NtPLR gene sequence in tobacco was amplified by PCR cloning technology,and the cloned NtPLR gene sequence was introduced into the expression vector PET28a through the construction of prokaryotic expression vector,and the expression protein was successfully induced.and the expressed protein was successfully induced and purified.In vitro enzyme activity assay of NtPLR was carried out using a spectrophotometer to determine that PL reacted under the action of PL reductase to generate PN.In this experiment,RNAi technology was used to construct RNAi vector containing NtPLR gene in tobacco.The down-regulation of NtPLR gene after 72h infection was best by fluorescence quantitative PCR.The down-regulation of NtPLR gene indirectly caused the general down-regulation of other VB6 metabolic enzyme gene transcription levels.In this study,agrobacterium-mediated transient expression of tobacco leaves was used to construct the NtPLR-GFP localization vector,which was shown to be located in the chloroplasts of cells under the laser confocal fluorescence microscope.