Research On Pyridoxal Kinase And Pyridoxine Phosphate Oxidase Gene By RNAi In Silkworm, Bombyx Mori

PLP is the primary coenzyme form,participating in the amino acid,glycogen,neurotransmitter,sphingomyelin,hemoglobin and nucleic acid synthesis and metabolism.and regulate the dynamic equilibrium of cells in the mammals.Due to the lack of PLP can cause serious neurological disease,PLP concentration is too high will produce toxic effects,resulting in motor and sensory nerve obstacles,thus regulating the synthesis and balance supply PLP is VB6 nutrition core issues.Currently PLP synthase structure studies have thorough,but the synthetic regulation is unclear.Studies have found that over-expression of transferase,the content of PLP increased along with,suggesting that in vivo PLP and PLP-dependent enzymes are dynamic equilibrium relationship.Silkworm secrete a great many silk,protein metabolism is vigorous,PLP as transaminase coenzyme,PLP demand increased.In order to further explore the PLP biosynthesis regulate and dynamic balance,We used silkworm as experimental material,down regulationt the PLK and PNPO gene expression by RNAi,and used fluorescence quantitative method to detect PLK and PNPO gene expression and change of transaminase gene transcription level.We analysised silk gland gene transcriptome by high-throughput sequencing methods after RNAi-treated,revealed PLP synthesis and regulation of gene regulatory networks associated with it.The results are as follows:1.PLK and PNPO gene RNAi study in silkworm(1)PLK and PNPO gene RNAi efficiency was achieved at 48 h after injection fragment si RNA(2)PLK best interference fragment is K-si RNA1,PNPO best interference fragment is o-si RNA2.(3)After PLK and PNPO gene RNAi treatment,the best tissues interference effects is midgut,the gene expression levels were decreased by 55% and 52%.Fat body almost no interference efficiency.2.Transaminase expression analysis after PLK and PNPO gene RNAiAfter PLK gene RNAi treatment,phosphoserine transaminase(phosphoserine aminotransferase,Ser B)and aspartate aminotransferase(asparate aminotransferase,AST)expression levels were decreased by 90% and 29% in silk gland;after PNPO gene RNAi treatment Ser B and AST gene expression levels were decreased by 90% and 29% in silk gland.3.Transcriptome analysis after PLK and PNPO gene RNAi in silkworm silk gland(1)After PLK gene RNAi treatment,365 differentially expressed genes were screened,of which 177 genes were down-regulated,188 genes were up-regulated.We analysised different gene by GO enrichment,found 156 Go terms which were significantly enriched(P?0.05),including 100 biological processes,cellular components 13 and 38 molecules Features.We analysised KEGG metabolic pathways,found 10 passages which were enrichment,differentially expressed genes mainly enriched protein processing in the endoplasmic reticulum,synthesis and accumulation of secondary material and protein export pathway.(2)After PNPO gene RNAi treatment,381 differentially expressed genes were screened,of which 260 genes were down-regulated,121 genes were up-regulated.We analysised different gene by GO enrichment,found 280 Go terms which were significantly enriched(P?0.05),including 140 biological processes,cellular components 65 and 75 molecules features.We analysised KEGG metabolic pathways,found 10 passages which were enrichment,differentially expressed genes mainly enriched protein processing in the the endoplasmic reticulum,protein output,ribosomes and amino acid biosynthetic pathway.This study demonstrates the silkworm PLP synthase gene linkages with aminotransferase gene regulation,silkworm protein synthesis was affected by PLP synthase RNAi.This study makes the foundation for further analysis of the work of PLP metabolic homeostasis and VB6,for the study of human nutrition issues VB6.