Research On The Function Of Silkworm GTP Cyclohydrolase Ⅰ(GTPCH) Gene In Melanin Synthesis And Metabolism

GTP Cyclohydrolase I(GTPCH, EC3.5.4.16)is a protease, which contains GTP-cyclohydro structure domain, and widely exists in vertebrates and invertebrates.GTPCH protease is an rate-limiting enzyme for reaction which finally forms tetrahydropterin(BH4) using GTP as substrate. In mammals, BH4deficiency will cause phenylketonuria, depression, dopa-responsive dystonia, dementia, hypertension, diabetes, atherosclerosis and other diseases. In insects, Drosophila GTPCH gene mutations will affect embryo melansim and hardening to death, Papilio xuthus GTPCH gene is related to formation of black stripe on larva integument. There is evidence that GTPCH enzyme activity in silkworm partridge spot(quail)mutant indviduals is higher than normal individuals, but has not been an report so far that directly concerns the role of GTPCH gene in silkworm melanin synthesis. Silkworm as Lepidoptera mode insects, studying the function of GTPCH gene in melanin synthesis and metabolism has great sigificance for revealing molecular mechanisms in silworm melanin synthesis and metabolism and exploring new way to prevent and control agricultural pests and diseases, at the same time pvoviding reference for the human disease control and treatment.Based on the silkworm genome database and by biological information analysis, RT-PCR, in vivo injection, tissue culture in vitro and HPLC techniques, the paper explores the function of GTPCH’in silkworm melanin synthesis and metabolism. The main results are as follows:1. BmGTPCH gene biological information analysis and expression researchSequence analysis showed:BmGTPCH amino acid sequence has supreme94per cent homology with Papilio xuthus, lower73per cent by Tribolium castaneum, lowest48per cent by human. GTPCH amino acid homology conse rved sequence among different species locates in GTP-cyclohydro functional domain.Chip data analysis showed:GTPCH gene has high quantity expression in gonads of day3of5th instar silkworm, a certain expression in head, silk gland, blood and fat body, a small amount of expression or no expression in epidermis, midgut and Malpighian tubules. This result is relatively similar to expression patterns obtained by phenylalanine hydroxylase (PAH)gene chip data analysis.RT-PCR results showed:In tissues of day3of5th instar silkworm larva, GTPCHa has expression in head tissues, not been found expression in blood, midgut, Malpighian tubules, midgut, silk gland, testis, ovary, trachea, fat body epidermal and so on, whose expression patterns are highly similar to TH; GTPCHb has high quantity expression in fat body, relatively obvious electrophoresis expression in head, testis, ovary, blood and other organizations, few quantity expression in Malpighian tubules, not been found expression in trachea, whose expression patterns are highly similar to PAH. During dormancy period of4instar, GTPCHa has expression on dormancy12h,18h,24h, not been found expression on dormancy Oh and6h, whose expression patterns are highly similar to TH; GTPCHb has expression on5development period after domancy,whose expression patterns are almost similar to PAH. A similar expression pattern suggests that GTPCH might be associated with the function of TH and PAH.2. Effect of GTPCH inhibitors on silkworm larva marking melanin synthesisMaking use of features such as forming new skin in different instar domant silkworm larva, newly synthesizing melanin which distribute on new skin and form natural body color markings, as well as more melanin in general dapple of larva and convenience of observation and comparison that dapple symmetrically distribute on the dorsal midline left and right sides, GTPCH enzyme specific inhibitors are injected in vivo into right dapple of3instar or4instar larva whose chest has entered the enlargement, changes in dapple of larva back after molting are observed, compared with left dapple of same individual without injection, there are positive ontogeny whose right dapple is shallow (dapple paler coloration), dwindle colored scope(dapple deletion), even not colored (dapple completely disappeared), the positive rates in6repeated trials under2injection concentration all reached more than85per cent. This result inhibition of GTPCH activity will affect the nomal melanin synthesis, ie. GTPCH activity is related to silkworm melanin synthesis.3. A slept silkworm integument tissue culture method convenient to study pigment metabolismTaking the integument tissue of4instar after sleeping as experimental materials, the paper explores larval integument culture method convenient to study silkworm pigment metabolism. The results showed:Grace medium with10%fetal bovine serum is suitable for larval integument tissue culture, the culture temperature is26℃; it is more easily to observe changes in pigment metabolism appearance for integument tissue in dapple removed old epidermal after sleeping11～12h, after24h incubation, the seta regeneration and pigment deposition is easy to be observed as marker trait in pigment metabolic changes. By the experiment of melanin synthesis inhibition in vitro observing appearance changes in integument, the paper demonstrate it is an important reaction step in melanin synthesis and metabolism of silkworm that tyrosine hydroxylase catalyse tyrosine into dopa.4. Molecular mechanisms in action of GTPCH on silworm melanin synthesisThe paper cultured and observed the integument tissue in dapple removed old epidermal after sleeping11～12h in4instar, the results showed:(1)melanin failed to form in integument with medium added GTPCH inhibitors, this again proved suppression of GTPCH enzyme activity can suppress melanin synthesis, namely GTPCH participates in silkworm melanin synthesis.(2) melanin formed in integument with medium added GTPCH inhibitors and BH4, namely under the condition that GTPCH is inactive, addition of BH4can make melanin synthesis and metabolism normal, it showed:The role of GTPCH in the process of silkworm melanin synthesis is to provide BH4or participate in melanin synthesis and metabolism by providing BH4.(3) melanin failed to form in integument with medium added GTPCH inhibitors and phe or added GTPCH inhibitors and tyr, while melanin formed in integument with medium added GTPCH inhibitors and dopa, namely under the condition that GTPCH is inactive, melanin can’t be derived from phe and tyr, but dopa, or under the condition that GTPCH is inactive, the reason that melanin can’t be derived from phe and tyr is dopa can’t be derived from phe and tyr, therefore, dopa derived from phe and tyr need BH4.(4) melanin failed to form in integument with medium added GTPCH inhibitors and PAH inhibitors and BH4, namely on condition that BH4is provided, making PAH inactive, preventing phe from converting into tyr, melanin synthesis cann’t be normal, it indicates phe is an important precursor for silkworm melanin synthesis, phe transform into tyr is an important step in silkworm melanin synthesis and metabolism; By detection of HPLC, the tyr content of integument with medium added GTPCH inhibitors and TH inhibitors and BH4is higher than integument with medium added GTPCH inhibitors and TH inhibitors, tyr metabolism and consumption is inhibited and melanin failed to form in two kinds of mediums, difference is whether or not BH4is provided in medium, tyr content is higher with BH4, high content showed there is the synthesis of tyr, therefore, the synthesis of tyr needs BH4, or phe transform into tyr needs BH4.(5) melanin failed to form in integument with medium added GTPCH inhibitors and PAH inhibitors and tyr, while melanin formed in integument with medium added GTPCH inhibitors and PAH inhibitors and tyr and BH4, the synthesis of endogenous tyr is inhibited and external tyr is provided in two kinds of mediums, difference is whether or not BH4is provided, melanin formed with BH4, therefore, melanin derived from tyr needs BH4, or tyr transform into dopa needs BH4. Comprehensively analysed the results of above kinds of culture:GTPCH participates in silkworm melanin synthesis and metabolism, GTPCH participates in silkworm melanin synthesis and metabolism by providing BH4, moreover,GTPCH participates in silkworm melanin synthesis and metabolism by providing BH4for two reaction steps that PAH catalyze phe into tyr and TH catalyze tyr into dopa.