Gene Expression Analysis Of Pyridoxal Kinase In Bombyx Mori

Vitamin B6 is a group of water-soluble vitamin and the generalterm for a glass of pyridine compounds, including the free forms of pyridoxal(PL),pyridoxine(PN), pyridoxamine(PM) and the phosphate forms of pyridoxal-5'-ph-osphate(PLP), pyridoxine-5'-phosphate(PNP), pyridoxamine-5'-phosphate(PMP). PLP is an important coenzyme of the amino acid metabolism,involving amino acids participate in catalysis of metabolic reactions. Pyridoxal kinase (PLK,) is a key enzyme about VB6 metabolism, as presence of Zn2+ and ATP, catalyzes PL, PN and PM generating PLP, PNP, PMP, in addition, PLK with pyridoxine 5'-phosphate oxidase, phosphatases maintain stabilization of PLP concentration in cells.In prior study, we have cloned cDNA (GenBank accession number:DQ452397) of Bombyx mori PLK from fat body of larva adopting bioinformatics principle and PCR method, and then constructed fusion expression vector pET22b-His-PLK, expressed in E.coli Rosetta and then purified recombinant protein, and analyzed characterization of PLK of Bymbox mori and protein structure was predicted.In this study, Fusion vector was expressed solvable recombinant protein in E.coli Rosetta, purified by Ni using affinity chromatography. The purified fusion protein was used to immunize a male New Zealand rabbit. Then, antiserum was harvested, and IgG was prepared by affinity chromatography using immobilized protein A. Some tissues of the fifth instar larvae, such as head, epidermis, silk gland, fat body, Malpighian tubules, .midgut, testis, ovary, were extracted and analyzed by Western blotting. The result showed that PLK existed in larva's testis, malpighian tubules, midgut, epidermis, head on the decrease. However, none was detected in larva's silk glands.We also compared the quantity of PLK mRNA in different tissues of the fifth instar larva of silkworm. These results suggested that PLK selectively expressed in silkworm's testis, malpighian tubules, head and midgut. By mRNA and protein levels from the study PLK gene in silkworm larvae at different developmental stages and different tissues of transcription, expression, .we definite the function of PLK in different tissues of the fifth instar larva, and this provides an important basis for the further study of the biological function of PLK in the time of the VB6 metabolism in the silkworm.Bombyx mori that is biological basis of. supporting silk industry, which is model organism is only second to drosophila. Furthermore, it is material of developing new bioreactor and industry of insect. The biosynthesis of amino acids is active in synthesis of silk protein and development of B.mori, VB6 is an important role. PLK may be a key point in the interconversion of VB6 in B.mori. The study about expression of PLK for clarify mechanism, expression and regulation of Bombyx mori is important.