The Study On The Expression And Function Of Tyrosine Hydroxylase Gene In The Silkworm, Bombyx Mori

Tyrosine hydroxylase (TH, E.C.1.14.16.2) is an enzyme that catalyze the hydroxylation of tyrosine. As the result, tyrosine become dopa. As a highly conservative enzyme in Vertebrates and invertebrates, tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis, which exist widely in insects, mammals and humans and is an essential metabolic enzyme. At present, TH has been reported in Human, Drosophila, Manduca sexta and papilio xuthus, but not yet in silkworm. With the post-genome era coming, the silkworm that is a lepidopteran model insect has become an ideal resource for gene functional studies. Besides, the silkworm genome data and gene chip data has been published, all of these provide the information platform for studies of silkworm genes. This study bases on the silkworm genome data and gene chip data, via bioinformatics, expression pattern, in situ hybridization, RNAi and determination of enzyme activity studies on Bombyx mori Tyrosine hydroxylase (BmTh) which is the key gene of melanin synthesis path way. The main results are as following:1 Bioinformatics analysis of BmThAnalyzing BmTh based on the data from NCBI and silkworm genome database. BmTh is 2272 bp long, contains 8 exons and 7 introns. The ORF is 1686 bp which encodes 561 amino acids. Anticipated weight of the protein is 63.4 kDa, PI 5.33.BmTh locates on chromosome 1,nscaf 1690. The analysis of protein domain indicated that Bombyx mori(BmTh), Drosophila melanogaster (DmTh), Papilio xuthus(PxTh) and Manduca sexta (MsTh) contain hydrogen biopterin domains. Phylogenetic analysis showed that, BmTh is close to that of other Lepidoptera insects and far away from that of mammals. 2 Expression pattern of BmThFirstly, the expressions of BmTh during embryo development from the 1st to 10th days were detected by RT-PCR. BmTh has no expression from the 1st to 5th days after pickling, it expressed lowly at the 6th day, but highly from 7th to 10th days.Secondly, the microarray data of development from 4th fifth instar larvae to adult showed that the expression level of BmTh has two peaks(the 60 hours after mounting and the stage before becoming moth). In the end,according to the silkworm microarray data, BmTh was found expressed most highly in the head of 3rd fifth instar larvae. It also expressed in the skin, but expressed low or no in other tissues. The results of RT-PCR are consistent with the results of microarray analysis.3 In Situ hybridization of BmThIn order to investigate the expressions of BmTh in different tissues, specific RNA probes were generated and performed in situ hybridization of paraffin section. Using the anti-sense chain of RNA as probe and sense chain as control, the rim of silkworm head and the inside new epiderm showed green florescence. This result confirmed the results of microarray and RT-PCR,and showed directly the positions of tissues expression of BmTh.4 RNA interference of BmThIn order to research the function of BmTh,silkworm embryos were injected with different concentrations of BmTh-dsRNA.The results indicated that after injecting BmTh-dsRNA,the colour of embryos head and body became brown and lost the ability of hatching.The varieties of BmTh mRNA were detected by real-time PCR. The results indicated that the expression of BmTh decreased significantly after injecting high(1000 ng/μL) concentration of BmTh-dsRNA compare with low(100 ng/μL) concentration. We presume that BmTh participate in the process of embryos melanin synthesis.Silkworm were also injected with tyrosine hydroxylase inhibitor (3-IT) at mounting stage, the result showed that injecting of 50 mM 3-IT affected the process of pupa.It supposes that the inhibitor restrained the activity of BmTh, which lead to larvae epiderm not enough hardening and abnormal metamorphism.Injecting of 100 mM 3-IT caused death and made the color of larvae turn black. It is possible that injected dose justo major,which lead to silkworm poisoning and darkening.5 Activity analysis of BmTHDopa is the product of the reaction between BmTH and tyrosine. The activity of BmTH under different temperatures were detected according to the yield of dopa by HPLC. This research investigated the activity of BmTH from 22℃to 40℃,each 3℃as an interval.The results indicated that 31℃-34℃are the most suitable temperatures for BmTH. Under those temperatures, there was the most amount of dopa and BmTH showed the highest activity. The results of injecting 3-IT into larvae showed that the activity of BmTH was inhibited after injecting 100 mM 3-IT more than 50 mM, which led to the decreasing of dopa yield.According to those results and the functions of tyrosine hydroxylase in other insects, BmTh plays an important role in controlling melanin synthesis and exoskeleton hardening, and is an indispensable key gene during the development of silkworm.