In Vitro And In Vivo Expression Of BmDhfr And Functional Analysis Involved In BH₄ Salvage Synthesis Pathway Of Silkworm Bombyx Mori

Tetrahydrobiopterin(BH4)is an essential cofactor for aromatic amino acid hydroxylase,nitric oxide synthase and alkylglycerol monooxygenase so that plays a key role in many biological processes.BH4 deficiency or disorder metabolism is associated with numerous neuropathic disease.Mammal studies clearly showed that there are four pathways in BH4 biosynthesis,including de novo synthesis pathways,salvage pathways,alternative pathways and regenerative pathways.Our previous studies have shown that the defective enzymatic activity of sepiapterin reductase(SPR)obstructed the de novo synthesis of BH4,causing the lemon mutant phenotype.When the other body color mutant inhibition lemon(i-lem)and lem is present at the same time,the body color of lem mutant larvae is obviously lightened,and whether the molecular mechanism of mutation is related to the synthesis and regulation of BH4 is unknown.Dihydrofolate reductase(DHFR)is one of the important enzymes involved in the salvage pathway.In order to understand the molecular function of BmDhfr in the regulation pathway of BH4 in silkworm,our study used ah09(lem),182(i-lem),RNAi-BmSpr transgenic silkworm and RNAi-BmSpr BmN cell lines as models to study obstructive de novo synthesis,investigating the mode of action and function of BmDHFR and its salvage pathway in the synthesis of BH4 in the silkworm body when the de novo synthesis pathway is blocked.Taking wild-type silkworm p50 as control,our experiment first analyzed the gene expression pattern of BmSpr and BmDhfr and the DHFR activity level in lem head tissue.The results showed that BmDHFR activity was up-regulated when BH4 synthesis was blocked.Then based on the study of gene expression patterns of BmSpr and BmDhfr of RNAi-BmSpr BmN cells and RNAi-BmSpr transgenic silkworm,and protein and enzyme activity levels of BmDHFR,the results show that BmDhfr mRNA,protein and enzyme were up-regulated when BmSpr expression was disturbed.It is predicted that when the de novo synthesis pathway of BH4 is blocked,the salvage pathway may play an active role in the silkworm.To further study the function of up-regulation expression of DHFR,we investigated the proteins expression levels and enzyme activities of BmDHFR in lem and i-lem mutant.The results showed that the BmDHFR protein and enzyme activity levels of i-lem were significantly up-regulated,indicating that the molecular mechanism of i-lem inhibit lem may be related to the salvage synthesis pathway of BH4.At the same time,the ratio of BH4 and dihydrobiopterin(BH2)of head,epidermis and fat body was up-regulated in i-lem.In view of the previous results of our experiments(the head,epidermis and fat body of lem had lower BH4 content than i-lem),we speculate that the salvage pathway is activated in i-lem mutant,which induces up-regulation expression of BmDHFR,thereby promoting the reaction of BH2 to produce BH4.The comprehensive results of this study indicate that BmDHFR and its salvage pathway plays an important role in maintaining the BH4 level,when BH4 de novo synthesis pathway is blocked in the silkworm.Our studies have shown that inhibition of BmDHFR activity down-regulate C1-tetrahydrofolate(BmC1-THF)synthase expression.BmCl-THF synthase plays a role in circulating tetrahydrofolate to 5-methyl tetrahydrofolate(5-MTHF),and 5-MTHF plays an important role in maintaining BH4 level.Therefore,BmDHFR may play an positive role in maintaining BH4 level by regulating BmC1-THF synthase expression.