Identification, Expression And Regulational Function Of Genes Involving In The BH4 Synthesis And Metabolism Pathways Of Bombyx Mori

Tetrahydrobiopterin(BH4) is an essential coenzyme of aromatic amino acid hydroxylases, as well as the cofactor of both nitric oxide synthase and alkylglycerol mono-oxygenase. In mammals, several neurological diseases are caused by BH4 deficiency. Previousstudies clearly showed that there were three pathways in BH4 biosynthesis. In the do novo pathway, BH4 were synthesized from GTP with three steps, catalyzed by three enzymes. BH4 were synthesized from the intermediatesepiapterin with two steps in salvage pathway. After catalytic reaction, BH4 were regenerated by the catalysis of two enzymes in the regeneration pathway. But there were still some unclearaspects on theregulatory networks of BH4 biosynthesis. The mechanism of the alternative pathways and the effect of their regulating factors were unknown, and the temporal and spatial expression profiles and functional status of different synthesis pathways were unclear. As a model lepidopteran insect, silkworm(Bombyx mori) has a clear genetic background. The draft and fine maps of the genome of domesticated silkworm have been completed, and information of silkworm databases were comprehensive. Several hundred of mutation strains were maintained in native and abroad, some of which were valuablebiological resources for the relateddiseases research in human. Yellow body color mutant lemon(lem), yellow body lethal mutant lemon lethal(leml) and albino lethal mutant albino(al) of silkworms are different mutations related to BH4 deficiency, which are excellent models to study on the synthesis and regulation of BH4. In this research, wild type silkworm strain Dazao(p50), different mutants related to BH4 deficiency and BmN cell line were used as materials, genes involved in BH4 synthesis were identified, expression and interaction pattern as well as their regulating roles were studied. The followings are the main results:1. Pterin-4α-carbinolamine dehydratase gene(BmPcd) and dihydropteridine reductase gene(BmDhpr)involving in the regeneration pathway of BH4 were cloned from the third day of fifth instar larvae. Multi-alignment of the amino acid sequences results showed that, both of the two genes showed a high homology to other related genes in other species, and showed a highest homology to those in fruit fly. The temporal and spatial expression patterns of BmPcd and BmDhpr were analyzed in different mutant strains. The results showed the expression pattern of the two genes were extremely different. The recombinant proteins of BmPCD and BmDHPR were expressed by the E.coli expression system, and enzymatic characters of BmDHPR were described. The recombinantBmDHPR showed a highest activity under room temperature and pH7, and exhibited different enzymatic parameters to the coenzyme of NADH and NADPH, suggesting that NADH was more suitable coenzyme than NADPH. rBmDHPRexhibited unsuitable parameter to the substrate, DMPH2, indicating rBmDHPR had a high specificity to substrate. The results of enzymatic activity in vivo between p50 and lemmutant ah09 showed that the regeneration pathway in B. moriwas priorly activated in the brain andsexual glands when the de novo pathway was blocked.2. 2-4 shRNAs were designedforthe sepiapterin reductase gene BmSprinvolving in the de novo pathway, dihydrofolate reductase gene BmDhfrinvolving in the salvage pathway, and BmDhprinvolving in the regeneration pathwayof BH4 synthesis, respectively, and related shRNA interferencevectors were constructed. The efficiency between two different transfectionreagent kits to BmN cell line was analyzed. The most effectiveshRNA interference vector for BmSpr and BmDhfr were selected by qRT-PCR. In the BmN cells, the relative expression levels of BH4-synthesis relating genes were not changed extremely when the expression of BmDhfr was down interfered, but when the expression of BmSpr was interfered, the relative expression level of BmPtps and BmDhfr were up-regulatedsignificantly. These results indicated that, the salvage pathway might take effect when de novo pathway was inhibited, and GTPCH and PTPS were the more importantrate-limiting enzyme compared to SPR in the de novo pathway of BH4. Moreover, the experiment of transgenic BmSpr-interferencemediated by shRNA was performed by microinjectioninto silkworm eggs.3. Total 17 transcriptsof 14 aldo-keto reductase genes(BmAr) and 9 transcriptsof 8 carbonyl reductase genes(BmCr) were found in the Chinese and Japanese silkworm genome databases and fulllength cDNA libraries using amino acid sequencesof mammalian AR and CR related to BH4 synthesis as queries by BLAST program. Phylogenetic analysis of the amino acid sequences of AR and CR proteins among different species showed that BmAKR12 and BmAKR13 exhibited highest homology with AR which has been reported as playing role in BH4 synthesis in mammals. Differential expression patterns of all the BmAr and BmCr predicted genes between p50 and ah09 strains were detected by semi-quantitative RT-PCR, by the results, BmAKR2 and BmCR3 were predicted as functional AR and CR members involving in BH4 synthesisin B. mori.In conclusion, the salvage pathway and regeneration pathway might have important biological functions in the BH4 synthesis and metabolism system of B. mori. The progresses made in this research arenecessaryexperimentalfoundationin biochemistry and molecular biology and contribute tocomprehensively understand the molecular regulationmechanism aboutBH4 synthesis and metabolism network in B. mori.Also, this paper provides a theoretical investigation for applyingthe BH4-deficiency associated mutants of B. morito be used as insect model for human diseases.