Cloning And Analysis Of Pyridoxine-5'-phosphate Oxidase And Polyamine Oxidase Genes In Tobacco

Vitamin B₆?VB₆?is a general term for a class of pyridine compounds.It is a colorless crystal that is easily soluble in water and ethanol.It is an essential vitamin for the human body.PLP is its main coenzyme form and is a coenzyme for many kinds of enzymes in the metabolism of organisms.Studies have shown that PLP is not only closely related to the metabolism of amino acids,but also participates in the regulation of cell signal transduction processes.The de novo synthetic pathway of VB₆ in nature is divided into two,one is the DXP-dependent pathway,which exists only in eukaryotic bacteria;the other is the DXP-independent pathway,and is the main pathway for the synthesis of VB₆ in nature.The six components of VB₆ can be converted to each other through metabolic pathways,among which PL kinase,PL reductase,PNP oxidase,pyridoxamine pyruvate transaminase and phosphatase play an important role in this process.The animals can not synthesize VB₆ by themselfs and can only convert the acquired VB₆ to the form required by the body through the metabolic conversion of VB₆.As a main source of animal acquisition of VB₆,it is of great significance to study the metabolic transformation of VB₆ in vivo.In this paper,PNPO and PAO were studied with model plant tobacco as material.1.Through the analysis of the known NtPNPO amino acid sequence,the NtPNPO sequence that removes the chloroplast transit peptide domain was cloned and called NtPNPO-2;meanwhile,the NtPNPOsequence thatremoved the chloroplast transit peptide domain and Yjef-N domain was called NtPNPO-3.The results showed that the NtPNPO-2coding frame was 1296 bp in length and encoded a protein containing 431 amino acid residues with a molecular mass of 48 KDa and isoelectric point is 6.31;the NtPNPO-3coding frame was 750 bp in length.It encoded a protein containing 249 amino acid residues with a molecular weight of 28 KDa and isoelectric point is 7.49.The fusion protein was successfully obtained by constructing Pmal-C2x-NtPNPO-2 and PmalC2x-NtPNPO-3.The results of enzyme activity assay showed that both NtPNPO-2and NtPNPO-3could catalyzed PMP.This suggested the Yjef-N domain is not an essential structure for the activity of PNPO.2.The polyamine oxidase cDNA sequence?called NtPAO?was obtained by comparison of the tobacco EST library,and the NtPAO was cloned to obtain the desired DNA fragment.The sequencing results showed that the length of the NtPAO coding frame was 1707 bp.It encoded a protein containing 568 amino acid residues with a molecular weight of 63 KDa and isoelectric point is5.35.Through the construction of Pmal-C2x-NtPAO prokaryotic expression vector,the target protein was successfully obtained.The results of enzyme activity assay showed that NtPAOcould catalyzed Spm.Subsequent enzymatic reactions using PM as a substrate showed that NtPAO can participate in the metabolic conversion of PM.