1 西南大学 生物技术学院，重庆 400715;
2 西南大学 家蚕基因组国家重点实验室，重庆 400716;
3 重庆大学 生命科学学院，重庆 401331
网络出版时间：2018-11-21
摘要: 5-羟色胺(5-hydroxytryptamine, 5-HT)是生物界广泛分布的信号分子，涉及动物的重要行为。5-HT是色氨酸羟化酶(Tryptophan hydroxylase, TRH)将L-色氨酸羟化为5-羟-L-色氨酸，5-羟-L-色氨酸随即被多巴脱羧酶(Aromatic L-amino acid decarboxylase, DDC)脱羧而成。TRH作为5-HT合成的限速酶，在无脊椎动物神经调控中具有重要地位。鳞翅目昆虫中TRH的功能研究并不多。在家蚕中克隆了家蚕TRH (Bombyx mori TRH, BmTRH)的cDNA序列1 667 bp，其中包含1 632 bp的开放读码框(Open reading frame, ORF)。人类TPH或者果蝇TRH (Drosophila TRH, DmTRH)与BmTRH有高度相似性，尤其BmTRH和DmTRH之间大多数氨基酸保守说明它们在系统发育上的密切关系并可能有相似功能。基因表达分析显示BmTRH主要表达于头部和中枢神经组织，免疫组织化学和Western blotting结果显示BmTRH只存在于神经组织中，即BmTRH可能仅参与家蚕的神经活动。此外，家蚕DDC (B. mori decarboxylase, BmDDC)和蛋白具有TRH活性的苯丙氨酸羟化酶基因(Phenylalanine hydroxylase, BmPAH)也在中枢神经系统中有表达，暗示家蚕神经系统5-HT的合成与果蝇中不同，可能有两种不同的调控机制。
关键词: 5-羟色胺 色氨酸羟化酶 分布 家蚕
Cloning and expression characteristics of tryptophan hydroxylase (TRH) from silkworm, Bombyx mori
Tian Li1, Xi Chen1, Haiyin Li1, Jiying Wang1, Wei Sun3, Qi Shen1, Cheng Lu2, Ping Chen1
1 College of Biotechnology, Southwest University, Chongqing 400715, China;
2 State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China;
3 Laboratory of Evolutionary and Functional Genomics, School of Life Sciences, Chongqing University, Chongqing 401331, China
Received: April 10, 2018; Accepted: August 6, 2018
Corresponding author:Ping Chen. E-mail: chenping1918@swu.edu.cn
Abstract: The biogenic monoamine 5-hydroxytryptamine (5-HT) is an ancient intracellular signaling molecule widely distributed in all animals with nervous systems, and has been implicated in principal behaviors. Tryptophan hydroxylase (TRH) induces a highly specific catalytic reaction that converts L-tryptophan (tryptophan) to 5-hydroxy-L-tryptophan (5-HTP) that is subsequently used as a substrate by aromatic L-amino acid decarboxylase (DDC) to form 5-HT. Five-HT is an ancient intracellular signaling molecule that is widely distributed in the animal kingdom and has been implicated in regulating the behaviors of animals with nervous systems. However, the role of TRH in Lepidoptera is not well understood. In this study, we cloned 1 667 bp cDNAs of Bombyx mori TRH (BmTRH), which contains a 1 632 bp open reading frame (ORF). Homology analysis revealed that BmTRH shared high amino acid identity with Homo sapiens TPH and Drosophila TRH (DmTRH). The high homology (70%) of BmTRH with DmTRH suggested that BmTRH could have a function similar to DmTRH. Gene expression analysis revealed that BmTRH was mainly expressed in head and central nervous (CNS). Moreover, immunohistochemistry and Western blotting analyses showed that BmTRH was detected only in larval nervous tissues. Taken together, our results indicate that BmTRH could likely function in the regulation of neural activities in B. mori. The transcripts of B. mori decarboxylase (BmDDC) and B. mori phenylalanine hydroxylase (BmPAH) whose proteins had TRH activity, were also expressed in the CNS tissues, indicating that unlike in Drosophila, two distinct mechanisms likely regulate 5-HT synthesis in silkworm.
Keywords: 5-HT Tryptophan hydroxylase distribution silkworm
IntroductionThe biogenic monoamine 5-hydroxytryptamine (5-HT or serotonin) is an ancient intracellular signaling molecule widely distributed in all animals with nervous systems, and has been implicated in various physiological functions and principal behaviors as a neurotransmitter modulator or a neurohormone. Five-HT was first discovered as a biogenic amine during gastrulation in the developing central nervous system (CNS) of mammals[1-2]. It was found to modulate various behaviors such as feeding, sleep, sexual behavior, body temperature, learning and memory[3-5]. In insects, 5-HT plays a crucial role in the regulation of salivary gland secretion, heart and oviduct contractions, circadian rhythms and diuresis[6-9]. In Drosophila, 5-HT was shown to play a vital role in early embryonic development[10-11], and in Manduca sexta, it was found to be indispensable for the development of olfactory glomeruli[12-14]. Previous studies in Bombyx mori have reported the role of 5-HT in the macroglomerular complex (MGC) and ordinary glomeruli in modulating the response of neuronal populations in the antennal lobe (AL)[15-16]. In summary, 5-HT is an essential compound during both behavioral and developmental processes[17].
Five-HT results from a cascade of reactions initiated by tryptophan hydroxylase (abbreviated as TRH in invertebrates and TPH in vertebrates), which is a rate-limiting enzyme that converts L-tryptophan (tryptophan) to 5-hydroxy-L-tryptophan (5-HTP). TRH is a member of the pterin-dependent aromatic L-amino acid hydroxylase family (AAAHs), which includes phenylalanine hydroxylase (PAH, EC 1.14.16.1) and tyrosine hydroxylases (TH, EC 1.14.16.2). Five-HTP is subsequently decarboxylated to generate 5-HT (a final product) by aromatic L-amino acid decarboxylase (DDC, EC. 4.1.1.28). TRH, as the rate-limiting enzyme, is known to regulate the concentrations of serotonin in vivo and has been reported to be more stable than 5-HT in insect neurocytes[18]. Therefore, TRH represents a specific property of 5-HTergic neurons, and evolutionary analyses have revealed that it is likely to have a broader role in the animal kingdom[19].
In vertebrates, TPH is encoded by two genes, and the two transcripts have tissue-specific patterns; TPH1 is expressed in the periphery while TPH2 is expressed in the CNS[20-23]. Studies in Drosophila melanogaster have revealed that the gene products of both PAH and TRH have tryptophan hydroxylase activity in vivo, and that TRH expressed in the neural tissues has a function similar to TPH2 in mice[24-27]. PAH, which primarily serves in phenylalanine hydroxylation, is expressed predominantly in the periphery and plays a role similar to TPH1 in rats[21]. Immunohistochemistry using sheep TPH antibody reveals the wide distribution of TRH protein in the brains of several insect species[25]. However, to our knowledge, there is no additional research on TRH in insects, particularly the order Lepidoptera, which includes important pests of agricultural crops. In this study, we cloned and characterized TRH from silkworm (a Lepidoptera model insect) to gain insights into its function in silkworm and other Lepidoptera.
1 Materials and methods1.1 Silkworm strainsSilkworm DaZao strain was maintained at the Southwest University in China, and fed on mulberry leaves under standard conditions (24–26 ℃ and 70%–85% RH with a photoperiod of 12:12 LD).
1.2 mRNA isolation and cDNA synthesisTotal RNA was purified using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. 3 μg RNA was reverse transcribed by Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega) to synthesize cDNA.
1.3 cDNA cloning of BmTRHcDNA from the heads of 5th instar DaZao larvae was used as templates in PCR for BmTRH amplification (the sequence of the primers in Table 1). PCR reaction conditions included 94 ℃ for 4 min, 30 cycles of 94 ℃ for 40 s, 55.5 ℃ for 1 min and 72 ℃ for 1.5 min and a final extension at 72 ℃ for 10 min. PCR products were purified and cloned into the pMD19-T simple vector (TaKaRa) and sequenced[28].
1.4 Multiple sequence alignment and phylogenetic analysisAmino acid sequences used for the sequence alignment were identified in the protein database of the NCBI with the amino acid sequence of BmTRH as template. Multiple sequence alignments of the amino acid sequences were performed with DNAMAN and ClustalX. Transmembrane spanning domains were predicted by TMHMM (genome.cbs.dtu.dk/services/TMMM). Phosphorylation sites and N-glycosylation sites were predicted by NetPhos (www.cbs.dtu.dk/services/NetPhos) and NetNGlyc (www.cbs.dtu.dk/services/NetNGlyc) respectively. Values for identity (ID) and similarity (S) were calculated by BioEdit. We utilized MEGA 6.0 to calculate the genetic distances among different species and to construct neighbor-joining (NJ) trees with 1 000-fold bootstrap resampling.
1.5 Semi-quantitative RT-PCR analysisAccording to the predicted CDS and EST sequences in the SilkDB, primers for expression analysis were designed for BmPAH, BmTRH and BmDDC (the sequences of the primers in Table 1). Conditions of PCR consisted of 94 ℃ for 4 min, 25 cycles at 94 ℃ for 40 s, 53 ℃ for 40 s and 72 ℃ for 1 min and a final extension at 72 ℃ for 10 min. Templates for the reaction were cDNA from eleven larval tissues (head, fat body, silk gland, tracheae, central nervous, hemolymph, testis, ovary, integument, malpighian tubule, midgut) from the 3-day-old 5th molting stage. BmActin3 was used as an internal control (the sequences of the primers in Table 1).
表 1 本研究所用的引物序列Table 1 The primer sequences used in this study
| Cloning (5′–3′) | Gene expression profiles (5′–3′) | Protein expression (5′–3′) | ||
| BmTRH | F | ATTTGGACGCAATGAGTGGT | ACCCAATACATTCGTCACTCGTC | GGATCCAGTGGTTCGGGAAAAGGTCTTCTA |
| R | CGTTTCATGAGCGTAATTTCGATAT | ATGCACAAATCTCCTCGTAACTC | CTCGAGTCACACCCACTGACCAGTATG | |
| BmPAH | F | AGTGTTCCACAGCACCCAGTA | ||
| R | TTGTCCATAGCGTTTAGCAG | |||
| BmDDC | F | GCTAAAATCACTACAGCCAGAC | ||
| R | GTTTATACGGCGTAATAGTTCTT | |||
| BmActin3 | F | TTCGTACTGGCTCTTCTCGT | ||
| R | CAAAGTTGATAGCAATTCCCT |
表选项
1.6 Recombinant protein expression and purificationThe coding sequence of BmTRH was amplified by PCR using head cDNA template (the sequence of the primers in Table 1). After digested with BamHⅠ and XhoⅠ(TaKaRa), the PCR product was ligated into pET-28a vector. Following with transformation into competent E. coli Rosetta (DE3) cells, and induced with 0.6 mmol/L isopropyl-β-D-1- thiogalactopyranoside (IPTG) for 6 h at 37 ℃ before protein extraction. The recombinant protein containing the 6×His tag was purified by affinity chromatography using a Ni2+ column. The purified protein was then quantified using the Bradford method.
1.7 Mass chromatographic analysis and antibody preparationPurified protein was analyzed by MALDI-TOF- MS as described previously[29]. Data were primarily downloaded from the silkworm genome database (http:silkworm.Swu.edu.cn/silkdb) and data from the NCBI protein database was used as a supplement. Peptide mass fingerprinting was analyzed by GPMAW software. The parameters used were as follows: precision = 0.10%; min. prec = 0.50 Da; min. hits =2; max. overlap = 2. Identification criteria were based on the number and coverage of matched peptides: a minimum of 5 peptides were required to match and the coverage of the matched peptides was about 25%.
A rabbit polyclonal antiserum was prepared against the recombinant BmTRH (Zoonbio, China). Immunizing a healthy male adult rabbits named New Zealand White and the rabbit was immunized every two weeks (three times more). Then collecting blood to gain the antiserum and finally the antibody titer is measured by ELISA (Sigma) and was 1: 6 000.
1.8 Western blotting analysisThe proteins of fat body, silk gland, tracheae, hemolymph, testis, ovary, integument, malpighian tubule and midgut were extracted from 3-day-old 5th Dazao strain. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 12% gels. After determined with the BCA protein assay, approximately 10 mg total protein was loaded per well. Proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) with a Mini-ProteinⅡblotting system (Bio-Rad) 200 mA, 100 min at 4 ℃ in a buffer containing 15% methanol. Membranes were blocked with 5% dry milk in Tris-buffered saline containing Tween 20 (TBS-T; 10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.01% Tween 20) for 30 min at room temperature. Membranes were probed with affinity-purified anti-BmTRH antibodies (dilution 1: 10 000 in TBS-T) and antigen-preabsorbed affinity-purified antibodies at the same concentration. Membranes were washed with TBS-T, followed by incubation with a secondary antibody conjugated to horseradish peroxidase (1: 2 000; Beyotime, China). Signals were visualized with an enhanced chemiluminescence detection system (ECL; Thermo, USA) and photographed using a Clinx ChemiScope 3400 Mini (China Scientific, China). Experiments were repeated for three times with independently isolated protein samples.
1.9 Immunohistochemistry assayThe testis, ovaries, integument and nervous of 3-day-old 5th instars from the silkworm Dazao strain were moved, and rapidly fixed with 4% paraformaldehyde for 2–4 h at room temperature. Samples were embedded in paraffin following dehydration (a sequential ethanol series: 30%, 50% and 70%, 100%). All the materials were treated vertical embedding. When cooled, samples were continually sectioned with the aid of paraffin section technique and the thickness of slices was all 5 μm. Paraffin sections were deparaffinized in xylene, rehydrated through graded ethanol solutions, quenched by 10 min immersion in 3% hydrogen peroxide, and treated with 0.01 mol/L citrate buffer (pH 6.0) at 92–98 ℃ for 15 min. Sections were incubated for 1–2 h in 10% goat serum at room temperature, and then incubated with the BmTRH antibody (dilution 1: 10 000) overnight at 4 ℃. After rinsing Tween-20-phosphated-buffered saline (TPBS), sections were incubated for 1–2 h with an HRP-labeled goat anti-rabbit antibody (dilution 1: 2 000) at room temperature. The sections were developed using DAB (3, 3-diaminobenzidine) and observed under a microscope. Serum of goat injected with PBS (negative serums) served as a negative control.
2 Results2.1 BmTRH cloning and expressionWe identified three potential TRH silkworm homologs by performing homology searches of the silkworm database (provide URL) using the nucleotide sequences of tryptophan hydroxylase from H. sapiens and D. melanogaster as query. Among the three, phenylalanine hydroxylase (BmPAH)[30] and tyrosine hydroxylase (BmTH)[31] have been previously reported. BmTRH, which had the highest homology with DmTRH and TRP, spans 10.31 kb and is located on nscaf1690 in chromosome 1. We cloned the 1 667 bp cDNAs sequence of BmTRH containing the 1 632 bp ORF (KF650639) from the heads of silkworm larvae by using RT-PCR (Fig. 1). BmTRH gene contained 10 exons and 9 introns (Fig. 2A), and encoded a putative protein containing 543 amino acids (aa) with an expected molecular weight of 62.15 kDa. To investigate of BmTRH expression profiles, the complete coding sequence of BmTRH was expressed in a prokaryotic expression system. SDS-PAGE analysis showed that the molecular weight of the expressed recombinant protein was about 62 kDa, which was consistent with the predicted weight of BmTRH (Fig. 2B, Fig. 3). Purity of the protein was ≥ 90% and it was identified as BmTRH by peptide mass fingerprinting (Fig. 2C). The peptide fingerprint masses were analyzed as GPMAW, which showed 20.1% coverage of matched peptides and 8 matched peptides (average difference = 0.49). These results indicated the successful in vitro expression of BmTRH.
 |
| 图 1 Bm TRH的克隆和表达鉴定 Fig. 1 The cloning and expression of BmTRH. (A) The lane of BmTRH is about 1 667 bp for TA cloning. (B) The cloning fragment is inserted into expression plasmid pET-28a. |
| 图选项 |
 |
| 图 2 BmTRH基因结构和Bm TRH蛋白体外表达鉴定。(A) BmTRH基因结构。黑框代表外显子，横线代表内含子。(B)考马斯亮蓝染色12% SDS-PAGE后在约62 kDa大小处出现蛋白条带。(C) BmTRH蛋白质谱分析图。 Fig. 2 The structure of BmTRH and identification of BmTRH expressed in vitro. (A) The structure of BmTRH. Exons were represented by black boxes and the intron length was represented by lines. (B) 12% SDS-PAGE of proteins stained with Coomassie brilliant blue. The molecular weight of the recombinant protein was about 62 kDa as estimated. (C) Peptide mass fingerprint by MALDI-TOF-MS. |
| 图选项 |
 |
| 图 3 BmTRH原核表达及Western blotting检测 Fig. 3 The prokaryotic expression and Western blotting analysis of BmTRH. (A) The expression of BmTRH in prokaryotic expression system by 0.6 mmol/L isopropyl-β-D-1-thiogalactopyranoside on SDS-PAGE gels. Lane 1: marker. Lane 2: precipitate after induction. Lane 3: supernatant after induction. Lane 4: non-induced control. Lane 5: empty vector induction. (B) Specificity of anti-Bm TRH antibody tested on induced proteins. Immunization with anti- BmTRH antibody (1:10 000) used for Western blotting analysis using 3, 3′, 5, 5′-tetramethylbenzidine assay. A single band of ~62 kDa was detected Western blotting of 10 mg induced proteins. |
| 图选项 |
2.2 Sequences analysesAnalysis of the BmTRH nucleotide sequence (http://smart.embl-heidelberg.de/) revealed that the predicted BmTRH protein had all the typical features of the AAAH family: an N-terminal regulatory (ACT) domain (spanning 49–113 aa), a catalytic domain (spanning 147–478 aa) and a C-terminal coiled-coil region (spanning 465–476 aa) involved in tetramerization (Fig. 4). Comparison of the BmTRH amino acid sequence with the known sequences of Homo sapiens TPH (HsTPH) and Drosophila TRH (DmTRH), indicated that the sequences were conserved in the catalytic domain, while the C-terminus containing the regulatory domains diverged among the species (Fig. 4). More importantly, all residues that were identified as important for the structural and functional properties of (HsTPH) and DmTRH[27], were also conserved in BmTRH (Fig. 4). For example, the phosphorylation sites were located in residues S84, 89 and 300; the characteristic VLMYGS, GYLSP, F281, E313, A349 and Y352 were predicted to bind BH4; the residues H291, 312, 317 and E357 were iron binding sites; the conserved Y275, R297, H312, F353, F358 and S377 were tryptophan interaction sites; and the leucine zipper at the N-termini reported to be involved in protein multimerization (Fig. 4). These findings suggested the presence of most typical features of TRH in BmTRH. In addition, the amino acids sequence of BmTRH shared 70% identity with DmTRH, 61% with HsTPH1, 57% with HsTPH2, 52% with BmPAH, DmPAH and HsPAH, 46% with BmTH or HsTH, and 43% with DmTH (Table 2).
 |
| 图 4 BmTRH与人类TPH1，TPH2和果蝇TRH氨基酸多重序列比对结果 Fig. 4 Comparison of amino acid sequences alignment of BmTRH with Homo sapiens TPH1, TPH2 and Drosophila TRH. The alignment was generated using ClustalW alignment software. Identical residues were shown as white letters against black, whereas conservatively substituted residues were shaded. Residues implicated in iron binding were marked by a (▽), residues implicated in BH4 binding by a (▼), and residues associated with Leucine zipper by a thinner line. The ACT domains were marked by a bold line, while catalytic domain was marked by a red box. Tryptophan blinding sites were indicated above (●) and phosphorylation sites above (*). The accession numbers of TRH in relevant species in GenBank are listed in Table 3. |
| 图选项 |
表 2 家蚕TRH与果蝇(PAH、TH和TRH)、人类(PAH、TH和TPH)之间的氨基酸同源比较分析Table 2 Amino acid homology scores among Bombyx TRH with Drosophila (PAH, TH and TRH) and Homo (PAH, TH and TPH)
| Bombyx PAH | Bombyx TH | Drosophila PAH | Drosophila TH | Drosophila TRH | Homo PAH | Homo TH | Homo TPH1 | Homo TPH2 | |
| Bombyx TRH | 52 | 46 | 52 | 43 | 70 | 52 | 46 | 61 | 57 |
| The accession numbers of PAH, TH, TRH (TPH) in relevant species in GenBank are listed in Table 3. | |||||||||
表选项
表 3 本研究相关物种的PAH、TH、TRH蛋白GeneBank登录号Table 3 The accession number of PAH, TH, TRH in relevant species in this study
 | PAH | TH | TPH 1 | TPH 2 |
| Homo sapiens | NP_000268 | NP_954986 | NP_004170 | NP_775489 |
| Mus musculus | NP_032803 | NP_033403 | NP_033440 | NP_775567 |
| Gallus gallus | NP_001001298 | 1:NP_990136 2:XP_001235001 | NP_990287 | NP_001001301 |
| Anolis carolinensis | XP_003220930 | 1:XP_003214871 2:XP_003220929 | XP_003214791.1 | XP_00322119 |
| Danio rerio | NP_956845 | 1:NP_571224 2:NP_001001829 | a:NP_840091 b:NP_001001843 | NP_999960 |
| Caenorhabditis elegans | NP_495863 | NP_871903 | NP_495584 | |
| Apis mellifera | XP_623300 | NP_001011633 | XP_394674 | |
| Drosophila melanogaster | NP_523963 | NP_476897 | NP_612080 | |
| Tribolium castaneum | XP_967025 | NP_001092299 | XP_967413 | |
| Bombyx mori | NP_001274766 | NP_001138794 | NP_001296518 | |
| Acyrthosiphon pisum | XP_001945589 | XP_008182999 | XP_001952801 | |
| Lepeophtheirus salmonis | EMLSAG00000009260 | EMLSAG00000001561 | EMLSAG00000001300 | |
| Helobdella robusta | XP_009031440 | XP_009022498 | XP_009018167 | |
| Physcomitrella patens | AAAH: XP_001774942 | |||
表选项
2.3 Phylogenetic analysisTo explore the phylogenetic relationship between members of AAAHs, we constructed a neighbor joining phylogenetic tree with 51 genes from 13 species including 5 vertebrates [Homo sapiens, Mus musculus, Gallus gallus, Anolis carolinensis, Danio rerio] and 8 invertebrates [Tribolium castaneum (Coleoptera), Acyrthosiphon pisum (Hemiptera), D. melanogaster (Diptera), B. mori (Lepidoptera), Apis mellifera (Hymenoptera), Lepeophtheirus salmonis (Arthropoda, Crustacea), Helobdella robusta (Annelida, Clitellata) and Caenorhabditis elegans]. The AAAH of physcomitrella, a lower plant, was used as an out-group; this protein was reported not only to have PAH function but also TH function and/or TRH function. All AAAHs clustered into three distinctclades, suggesting that the members of AAAH which likely derived from an ancestor differed in their structures and/or function[32]. Moreover, the TRH clade was closer to the PAH clade than to the TH clade (Fig. 5). The first clade was the TH clade, which included two subclades, the invertebrate TH subclade comprised insect TH as well as other invertebrate TH, which served as the out-group while the vertebrate TH subclade consisted of TH1 and TH2 groups. Second was the PAH clade where vertebrate PAH and insect PAH belonged to different categories, and other invertebrate PAH were the out-group. Third was the TRH clade comprised the invertebrate TRH subclade and vertebrate TPH subclade. The invertebrate TRH subclade contained insect TRH groups where silkworm clustered together with D. melanogaster and other invertebrate TRH. The vertebrate TPH subclade was divided into TPH1 and TPH2 groups, revealing the likely difference in functions between TPH1 and TPH2. Furthermore, TPH2 was mainly expressed in the brain stem, while TPH1 was expressed in the gut, pineal gland, spleen, and thymus in human, mouse and rat[20]. Interestingly, TPH1 in Danio genome had two copies corresponding to gene duplication events that occurred specifically in teleost fish[33].
 |
| 图 5 基于氨基酸序列构建的无脊椎动物与脊椎动物TRH、TH和PAH系统进化树 Fig. 5 Phylogenetic tree of invertebrates and vertebrates TRH (a narrative convenience, is called vertebrate TPH and invertebrate TRH collectivity in here), TH and PAH. There were three distinct clades, TRH clade, TH clade and PAH clade, for all aromatic amino acid hydroxylases in animal. TRH, PAH and TH was indicated in pink, yellow-green and lake blue respectively. The accession numbers of PAH, TH, TRH in relevant species in GenBank are listed in Table 3. |
| 图选项 |
2.4 mRNA expression profileThe TRH activity product, 5-hydroxy-L-tryptophan (5-HTP) is derived from 5-hydroxytryptamine in a reaction that requires aromatic L-amino acid decarboxylase (DDC) that is also involved in the biosynthesis of dopamine[26]. We previously reported that the recombinant BmPAH protein had some TRH activity[30], suggesting that BmDDC, BmPAH and BmTRH could be related to serotonin synthesis. Thus, the tissue-specific expression patterns of BmTRH, BmDDC and BmPAH mRNA in eleven larval tissues (including head, fat body, silk glands, tracheae, central nervous, hemolymph, testis, ovary, integument, malpighian tubule and midgut) were investigated by RT-PCR. We found that the 536 bp BmTRH fragment was expressed only in the head and CNS with the PCR product being more intense in CNS than the head. On the other hand, a relatively bright band corresponding to 664 bp BmDDC product was detected in the head, fat body, tracheae, CNS, testis, ovary, integument and midgut, while the 567 bp PCR BmPAH fragment was observed in head, fat body, CNS, hemolymph, testis, ovary, integument and midgut. Weak PCR amplification was observed for BmDDC and BmPAH fragments in the hemolymph and malpighian tubule, respectively (Fig. 6 and Fig. 7). These results showed that BmTRH mRNA expression was highly restricted in space, and the transcripts of both BmDDC and BmPAH were expressed widely in most tissues, indicating their functional importance in various physiological contexts.
 |
| 图 6 BmTRH、BmPAH和BmDDC基因在家蚕幼虫各组织中的表达模式 Fig. 6 Expression profiles of BmTRH, BmPAH and BmDDC in larval tissues of silkworm. Lane 1; head; lane 2; central nervous; lane 3; integument; lane 4; fat body; lane 5; hemolymph; lane 6; silk glands; lane 7; midgut; lane 8; testis; lane 9; ovary; lane 10; trachea; lane 11; malpighian tubule; and control; actin control. All of the polymerase chain reaction products were approximately 600 bp. |
| 图选项 |
 |
| 图 7 家蚕幼虫各组织中BmTRH，BmPAH and BmDDC基因表达分析(泳道1：头；泳道2：中枢神经；泳道3：表皮；泳道4：脂肪体；泳道5：血淋巴；泳道6：丝腺；泳道7：中肠；泳道8：精巢；泳道9：卵巢；泳道10：气管；泳道11：马氏管。BmTRH，BmPAH and BmDDC基因PCR扩增产物大小分别是：536 bp，664 bp和567 bp) Fig. 7 Expression profiles of BmTRH, BmPAH and BmDDC in larval tissues of silkworm. Lane 1: head; lane 2: central nervous; lane 3: integument; lane 4: fat body; lane 5: hemolymph; lane 6: silk glands; lane 7: midgut; lane 8: testis; lane 9: ovary; lane 10: trachea; lane 11: malpighian tubule. The polymerase chain reaction products of BmTRH, BmPAH and BmDDC were 536 bp, 664 bp and 567 bp, respectively. |
| 图选项 |
2.5 BmTRH protein expression profileBmTRH was detected in larval tissues using anti-BmTRH antibody. Western blotting showed a specific band of ~62 kDa in the head and ventral chain of larvae, and high expression was observed in the ventral chain than the head. BmTRH was not expressed in fat body, silk glands, tracheae, hemolymph, testis, ovary, integument, malpighian tubule and midgut (Fig. 8). These results consistent with the mRNA expression profile of BmTRH.
 |
| 图 8 Bm TRH在家蚕幼虫各组织的Western blotting检测 Fig. 8 Western blotting analysis with the anti-BmTRH antibody in larval tissues of silkworm. Lane 1: tracheal; lane 2: ovary; lane 3: testis; lane 4: silk gland; lane 5: hemolymph; lane 6: fat body; lane 7: integument; lane 8: ventral chain; and lane 9: head. The specific lane was showed in lane 8 and 9. |
| 图选项 |
2.6 Localization analysis of BmTRH ImmunohistochemistryTo further analyze the expression of BmTRH, immunohistochemistry was performed on larval tissues embedded in paraffin sections. Clear positive signal was detected in CNS (ganglion at any region and several nerve cords containing brain), whereas weak signal was found in the integument, testis and ovary (Fig. 9 and Fig. 10). These results suggested that BmTRH expression was restricted to larval nervous tissues.
 |
| 图 9 Bm TRH在家蚕幼虫中枢神经，表皮，精巢和卵巢中的免疫组织化学分析 Fig. 9 Immunohistochemical analysis of BmTRH in central nervous, integument, testis and ovary of larvae. The arrows indicate positive signals in (A) (ventral ganglion) and (B) (brain). No positive signal was found in (C) (testis), (D) (ovary) or (E) (integument). Incubating with PBS displaced primary antibody was as negative controls. |
| 图选项 |
 |
| 图 10 Bm TRH蛋白在家蚕脑和腹部神经节的免疫荧光鉴定(Bm TRH抗体在脑和腹部神经节中均有表达) Fig. 10 Immunofluorescence analysis using BmTRH antibodie in ventral ganglion and brain of B. mori. BmTRH could be detected in ventral ganalion and brain. |
| 图选项 |
3 DiscussionIn this study, we identified only a single copy of the TRH gene in the silkworm genome, which is consistent with reports in other invertebrates. This is the first time of the cloning of its heterologous expression in a Lepidoptera insect. The BmTRH gene codes a 61.31 kDa protein consisting of 543 amino acids (aa), similar to DmTRH, which encodes a 61 kDa protein with 555 aa. BmTRH also has conserved residues sharing the structural and functional properties of HsTPH or DmTRH. These findings suggest that the BmTRH cloned here is a functional TRH.
In D. melanogaster, DmTRH exhibits high activity with a notable ability to hydroxylate phenylalanine, while DmPAH has strong phenylalanine hydroxylase activity and displays a significant ability to hydroxylate tryptophan. Both DmTRH and DmPAH have been reported, not to have tyrosine hydroxylase activity[24-26]. Our phylogenetic analysis indicates that TRH is closer to PAH than TH, and BmTRH together with DmTRH are clustered to a branch with TRH from other insect. Besides, our previous study also showed that BmPAH expressed in vitro was capable of tryptophan and phenylalanine hydroxylation rather than only tyrosine hydroxylation[30]. These results imply that BmTRH, like DmTRH, can hydroxylate both tryptophan and phenylalanine but not tyrosine in silkworm.
The BmTRH transcript and BmTRH protein are detected only in the head and CNS with high expression in the CNS. Consistently, immunohistochemistry also shows a strong positive signal only in CNS tissues containing the ventral ganglion and brain. These suggest that BmTRH could likely trigger neural activities in silkworm larvae, and is consistent with previous reports that TRH participates in the neural activity in other animals[34-35].
In D. melanogaster, DmPAH and DmTRH are selectively involved in 5-HT synthesis with distinct expression patterns and enzyme activities[36]. However, expression analysis in this study shows that BmTRH together with BmPAH and BmDDC could likely be co-expressed in the head and CNS tissues. These suggest the likelihood of two distinct regulation mechanisms for 5-HT synthesis in CNS, and that BmTRH- and BmPAH-mediated 5-HT biosynthesis pathways may not segregate into neuronal and peripheral tissues in silkworm. A similar phenomenon has been reported in Gryllus bimaculatus[37].
4 ConclusionIn this study, we identify and clone a cDNA sequence for TRH gene in silkworm, and analyze the phylogenetic relationships to metazoan as a members of aromatic amino acid hydroxylases (AAAHs). By gene and protein expression analysis, we speculate BmTRH could likely function in the regulation of neural activities. This is the first time that the BmTRH gene is cloned from silkworm and BmTRH protein is expressed and purified in vitro in Lepidoptera.
Acknowledgement
Thanks to Feifei Huang, Zhanpeng Dong and Zhouhe Du of Southwest University for providing technical support.
REFERENCES
| [1] | Lauder JM, Bloom FE. Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. 1. Cell differentiation. J Comp Neurol, 1974, 155(4): 469-481. |
| [2] | Lauder JM, Sze PY, Krebs H. Maternal influences on tryptophan hydroxylase activity in embryonic rat brain. Dev Neurosci, 1981, 4(4): 291-295. |
| [3] | Lucki I. The spectrum of behaviors influenced by serotonin. Biol Psychiat, 1998, 44(3): 151-162. DOI:10.1016/S0006-3223(98)00139-5 |
| [4] | ?gren SO, Eriksson TM, Elvander-Tottie E, et al. The role of 5-HT1A receptors in learning and memory. Behav Brain Res, 2008, 195(1): 54-77. |
| [5] | Veenstra-VanderWeele J, Anderson GM, Cook EH Jr. Pharmacogenetics and the serotonin system: initial studies and future directions. Europ J Pharmacol, 2000, 410(2/3): 165-181. |
| [6] | Lee PT, Lin HW, Chang YH, et al. Serotonin-mushroom body circuit modulating the formation of anesthesia-resistant memory in Drosophila. Proc Natl Acad Sci USA, 2011, 108(33): 13794-13799. DOI:10.1073/pnas.1019483108 |
| [7] | Maddrell SH, Herman WS, Mooney RL, et al. 5-hydroxytryptamine: a second diuretic hormone in Rhodnius prolixus. J Exp Biol, 1991, 156: 557-566. |
| [8] | Nagao T, Tanimura T, Shimozawa T. Neurohormonal control of the mating interval in the male cricket, Gryllus bimaculatus DeGeer. J Comp Phys A, 1991, 168(2): 159-164. |
| [9] | Neckameyer W, Bhatt P. Neurotrophic actions of dopamine on the development of a serotonergic feeding circuit in Drosophila melanogaster. BMC Neurosci, 2012, 13(1): 26. |
| [10] | Colas JF, Launay JM, Maroteaux L. Maternal and zygotic control of serotonin biosynthesis are both necessary for Drosophila germband extension. Mech Dev, 1999, 87(1/2): 67-76. |
| [11] | Colas JF, Launay JM, Vonesch JL, et al. Serotonin synchronises convergent extension of ectoderm with morphogenetic gastrulation movements in Drosophila. Mech Dev, 1999, 7(1/2): 77-91. |
| [12] | Mercer AR, Hayashi JH, Hildebrand JG. Modulatory effects of 5-hydroxytryptamine on voltage-activated currents in cultured antennal lobe neurones of the sphinx moth Manduca sexta. J Exp Biol, 1995, 198(3): 613-627. |
| [13] | Mercer AR, Kirchhof BS, Hildebrand JG. Enhancement by serotonin of the growth in vitro of antennal lobe neurons of the sphinx moth Manduca sexta. J Neurobiol, 1996, 29(1): 49-64. DOI:10.1002/(ISSN)1097-4695 |
| [14] | Oland LA, Kirschenbaum S, Pott W, et al. Development of an identified serotonergic neuron in the antennal lobe of the moth and effects of reduction in serotonin during construction of olfactory glomeruli. J Neurobiol, 1995, 28(2): 248-267. DOI:10.1002/(ISSN)1097-4695 |
| [15] | Hill ES, Iwano M, Gatellier L, et al. Morphology and physiology of the serotonin-immunoreactive putative antennal lobe feedback neuron in the male silkmoth Bombyx mori. Chem Senses, 2002, 27(5): 475-483. DOI:10.1093/chemse/27.5.475 |
| [16] | Hill ES, Okada K, Kanzaki R. Visualization of modulatory effects of serotonin in the silkmoth antennal lobe. J Exp Biol, 2003, 206(2): 345-352. |
| [17] | Monastirioti M. Biogenic amine systems in the fruit fly Drosophila melanogaster. Microsc Res Tech, 1999, 45(2): 106-121. DOI:10.1002/(ISSN)1097-0029 |
| [18] | Severson CA, Wang WG, Pieribone VA, et al. Midbrain serotonergic neurons are central pH chemoreceptors. Nat Neurosci, 2003, 6(11): 1139-1140. DOI:10.1038/nn1130 |
| [19] | Cao J, Shi F, Liu XG, et al. Phylogenetic analysis and evolution of aromatic amino acid hydroxylase. FEBS Lett, 2010, 584(23): 4775-4782. |
| [20] | C?té F, Thévenot E, Fligny C, et al. Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function. Proc Natl Acad Sci USA, 2003, 100(23): 13525-13530. |
| [21] | Patel PD, Pontrello C, Burke S. Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland. Biol Psychiat, 2004, 55(4): 428-433. |
| [22] | Walther DJ, Peter JU, Bashammakh S, et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science, 2003, 299(5603): 76. DOI:10.1126/science.1078197 |
| [23] | Yu PL, Fujimura M, Okumiya K, et al. Immunohistochemical localization of tryptophan hydroxylase in the human and rat gastrointestinal tracts. J Comp Neurol, 2009, 411(4): 654-665. |
| [24] | Alca?iz S, Silva FJ. Phenylalanine hydroxylase participation in the synthesis of serotonin and pteridines in Drosophila melanogaster. Comp Biochem Phys C: Pharmacol, Toxicol Endocrinol, 1997, 116(3): 205-212. |
| [25] | Bao XX, Tian XM, Zhao ZF, et al. Immunohistochemical evidence for the presence of tryptophan hydroxylase in the brains of insects as revealed by sheep anti-tryptophan hydroxylase polyclonal antibody. Cell Tissue Res, 2008, 332(3): 555-563. |
| [26] | Coleman CM, Neckameyer WS. Substrate regulation of serotonin and dopamine synthesis in Drosophila. Invertebr Neurosc, 2004, 5(2): 85-96. |
| [27] | Coleman CM, Neckameyer WS. Serotonin synthesis by two distinct enzymes in Drosophila melanogaster. Arch Insect Biochem Physiol, 2005, 59(1): 12-31. |
| [28] | Chen P, Wang JY, Li HY, et al. Role of GTP-CHI links PAH and TH in melanin synthesis in silkworm, Bombyx mori. Gene, 2015, 567(2): 138-145. DOI:10.1016/j.gene.2015.03.043 |
| [29] | Hou Y, Zou Y, Wang F, et al. Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages. Proteome Sci, 2010, 8: 45. DOI:10.1186/1477-5956-8-45 |
| [30] | Chen P, Li L, Wang JY, et al. BmPAH catalyzes the initial melanin biosynthetic step in Bombyx mori. PLoS ONE, 2013, 8(8): e71984. DOI:10.1371/journal.pone.0071984 |
| [31] | Liu C, Yamamoto K, Cheng TC, et al. Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm, Bombyx mori. Proc Natl Acad Sci USA, 2010, 107(29): 12980-12985. DOI:10.1073/pnas.1001725107 |
| [32] | Neckameyer WS, Holt B, Paradowski TJ. Biochemical conservation of recombinant Drosophila tyrosine hydroxylase with its mammalian cognates. Biochem Genet, 2005, 43(7/8): 425-443. |
| [33] | Dehal P, Boore JL. Two rounds of whole genome duplication in the ancestral vertebrate. PLoS Biol, 2005, 3(10): e314. DOI:10.1371/journal.pbio.0030314 |
| [34] | Nakamura K, Sugawara Y, Sawabe K, et al. Late developmental stage-specific role of tryptophan hydroxylase 1 in brain serotonin levels. J Neurosci, 2006, 26(2): 530-534. |
| [35] | Nakamura K, Hasegawa H. Developmental role of tryptophan hydroxylase in the nervous system. Mol Neurobiol, 2007, 35(1): 45-53. |
| [36] | Neckameyer WS, Coleman CM, Eadie S, et al. Compartmentalization of neuronal and peripheral serotonin synthesis in Drosophila melanogaster. Genes Brain Behav, 2007, 6(8): 756-769. DOI:10.1111/gbb.2007.6.issue-8 |
| [37] | Watanabe T, Sadamoto H, Aonuma H. Identification and expression analysis of the genes involved in serotonin biosynthesis and transduction in the field cricket Gryllus bimaculatus. Insect Mol Biol, 2011, 20(5): 619-635. DOI:10.1111/imb.2011.20.issue-5 |
