Genetic Regulation Analysis Of BH4 Synthesis In BmSPR-dificient Silkworm

Tetrahydrobiopterin(BH4),an important cofactor of many enzymes such as aromatic amino acid hydroxylase,nitric oxide synthase and alkyl glycerol monooxygenase,plays an indispensable role in living organisms.Sepiterin reductase(SPR)is the last enzyme in the de novo synthesis pathway of BH4.There is still a lack of clear understanding of the diagnosis and treatment of human BH4 deficiency caused by SPR mutations.A large number of studies in different animal models will help to fully understand this type of disease and improve the efficiency of diagnosis and treatment.Bombyx mori is a Lepidoptera insect model with a clear genetic background and abundant mutant strains.Previous studies in our laboratory have confirmed that lem and its recessive lethal lem~l(second instar silkworm hunger striker)are a BmSPR-deficient mutant.In this study,ah09(lem),RNAi-BmSpr,a65(lem~l)and wild type silkworm strains such as p50 and Nistari were used as experimental materials to analyze the genetic regulation of BH4synthesis.The following results were obtained:(1)Semi-quantitative RT-PCR was used to investigate the gene expression of BH4synthesis-related enzymes in the tissues of third day of fifth instar larvae in four strains other than a65.It was found that there were significant differences in the expression of the BH4 salvage synthesis pathway gene Bm Dhfr between different strains,indicating that the blocking of the de novo synthesis pathway may have a certain influence on the salvage synthesis pathway.(2)The content of BH4 in lethal individuals(leml/leml)was significantly lower than in wild-type larvae after first molt silkworm of a65 strain.The results of the administration of BH4 and monoamine(dopamine and serotonin)to the a65 larvae in vitro showed that BH4 has a significant therapeutic effect on lethal individuals.BH4 content and administration experiment showed that physiology reason of lethal individuals died is a serious lack of BH4 in the body.(3)The epidermis of a65 second instar silkworm(lem~l/lem~l)was observed by scanning electron microscopy and compared with wild type.The results showed that compared with the wild type individuals,the epidermis folds of the lethal individuals were more compactness,indicating that the lack of BH4 has certain effects on the degree of epidermis sclerotization and staining,which is consistent with previous estimates.(4)We measured BmSPR activity and content of BH4 in the fat body and gonads of a65 different genotypes(+/+or+/leml)moths before and after mating and oviposition.The results showed that the synthesis of BH4 was mainly in the fat body during moth stage.Most of the BH4 synthesized by the mother moth was transported to the ovary,however the testis contained almost no BH4.The content of BH4 in the ovary after mating and oviposition was significantly lower than that before mating and oviposition.The result indicated that BH4 in the ovary largely enters the fertilized egg,which is transmitted from the mother to the offspring.Thus,the growth and development of lethal individuals in the embryonic and first instar larval stages depends on the maternal synthesis of BH4,which can explanation for the maternal effects of the leml study.