【作者】 陈萍；

【导师】 鲁成；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2011， 博士

【摘要】 芳香族氨基酸羟化酶(AAAH)对芳香族氨基酸代谢具有重要的生理作用。芳香族氨基酸羟化酶家族(AAAHs)包括苯丙氨酸羟化酶(PAH)、色氨酸羟化酶(TRH)和酪氨酸羟化酶(TH)。苯丙氨酸羟化酶(PAH)广泛分布于动物、植物和微生物。苯丙氨酸羟化酶(PAH)是苯丙氨酸代谢的关键酶和限速酶,催化苯丙氨酸转变成酪氨酸,是哺乳动物体内苯丙氨酸正常分解代谢唯一途径的限速步骤。人类PAH基因与重要代谢疾病苯丙酮尿症(PKU)密切相关,线虫的PAH基因与表皮的黑色素合成有关,果蝇PAH基因与表皮硬化、眼色素合成以及神经活动有关,埃及伊蚊PAH基因与防御免疫黑化有关,但至今未见关于鳞翅目昆虫PAH基因的相关报道。家蚕作为鳞翅目昆虫代表,明确家蚕PAH基因(BmPAH)的作用和功能,对充分发挥家蚕的模式作用,在农林上建立防治鳞翅目害虫的新技术和新途径具有重要意义,同时对人类重要疾病PKU的治疗也具有参考价值。色氨酸羟化酶(TRH)是5-羟色胺(5-HT)合成的限速酶和关键酶,其活性的高低直接决定着神经元内5-HT的含量,色氨酸羟化酶(TRH)是确定5-HT能神经元的最好标志。5-HT广泛存在于脊椎动物和无脊椎动物的神经系统,参与多种行为和发育调节。临床上,TRH基因的多态性伴随多种心理障碍疾病,如抑郁症、强迫症、厌食症、食欲过旺症、老年前痴呆症以及精神分裂症等。昆虫上,关于TRH基因的研究报道很少,仅果蝇中有相关研究。家蚕作为模式生物,克隆和表达家蚕TRH基因(BmTRH)对推进识别昆虫5-HT能神经元的形态、类型、分布以及各神经元之间的联系具有重要意义。基于上述理由,本论文对家蚕PAH基因(BmPAH)和TRH基因(BmTRH)进行了克隆、表达及功能研究,主要结果如下：1、BmPAH基因克隆、序列分析及表达模式研究本研究克隆的BmPAH基因cDNA序列长1433bp,其中ORF序列长1371bp。BmPAH基因位于家蚕1号染色体nscaf2734上,包含6个外显子和5个内含子,外显子/内含子边界均符合GT-AG规则；BmPAH基因编码456个氨基酸,预测蛋白的分子量大小约为52kDa,等电点为5.46。预测显示BmPAH蛋白包含完整的ACT结构域和羟化苯丙氨酸的结构域,无信号肽序列,属非分泌蛋白。BmPAH基因组织表达分析结果表明：BmPAH基因转录本在家蚕5龄3天的精巢、卵巢、头、体壁和脂肪体中有高量表达,血液中有表达但不高,在丝腺、中肠、气管和马氏管中表达量低或不表达。芯片数据和RT-PCR实验结果完全一致。以家蚕个体总cDNA为模板,RT-PCR实验结果显示,BmPAH基因转录本在家蚕胚胎期表达量依胚胎发育大小而不同,发育时间长的大胚胎比发育时间短的小胚胎表达量高,这与家蚕胚胎后期合成大量黑色素比较一致；BmPAH基因转录本在家蚕幼虫各龄的眠蚕起蚕都有表达,与幼虫蜕皮合成大量黑色素、表皮重新着色比较一致；BmPAH基因转录本在家蚕变态期的表达量,总体上明显少于胚胎和幼虫两个阶段。2、BmPAH基因原核表达、western blotting及免疫组化分析本研究构建了序列和插入方向都正确的BmPAH基因原核表达载体——BmPAH/pET-28a(+)重组质粒。在原核中表达的蛋白出现于沉淀,经质谱鉴定的确为BmPAH基因编码的蛋白。用BmPAH基因原核表达蛋白免疫家兔获得的多克隆抗体进行免疫印迹和免疫组化实验。western blotting实验显示,家蚕幼虫5龄3天的脂肪体、卵巢和精巢3个组织总蛋白中有BmPAH蛋白的阳性信号,脂肪体中信号相对较强；BmPAH蛋白在脂肪体中大量表达,与家蚕脂肪体象人类肝脏、是苯丙氨酸代谢的主要场所一致。5龄3天幼虫组织的免疫组化结果显示,在家蚕头部整个组织都有BmPAH蛋白的阳性信号,与家蚕头部分布有黑色素、着生许多感觉器官一致；在体壁的真皮层和内表皮中均检测到BmPAH蛋白的阳性信号,与真皮细胞和内表皮是体壁黑色素等物质合成的场所相符；在精巢膜和卵巢膜上BmPAH蛋白信号较强,精巢膜内生精囊等也有目的蛋白信号,可能与家蚕生殖腺发育以及生殖细胞成熟有关；在中枢神经组织中BmPAH蛋白阳性信号较强,可能与神经活动有关。免疫组化实验结果与RT-PCR检测结果完全吻合,但与免疫印迹结果不一致,出现这种现象的原因可能是目标蛋白在总蛋白中含量太低以致免疫印迹检测不到引起。3、BmPAH基因在家蚕黑色素合成上的功能研究无论用自己合成的dsRNA片段还是公司生产的siRNA,注射产下3小时以内的蚕卵,都出现一定数量的着色浅、着色不均甚至不着色的发育但不孵化的阳性表型胚胎,荧光定量PCR检测结果均显示,阳性表型胚胎中的BmPAH基因转录本表达量明显低于对照,暗示出现的阳性表型与BmPAH基因被RNA干涉密切相关。HPLC-FLU分析siRNA注射的蚕卵,结果siRNA阳性个体中酪氨酸含量明显低于siRNA阴性个体和对照,且siRNA阳性个体中的苯丙氨酸有曾积累的痕迹,这与BmPAH基因被干涉,转录本表达量降低,基因产物量减少,催化反应活动变弱,形成酪氨酸的产物量降低一致。HPLC-FLU分析结果与荧光定量PCR检测结果能够相互印证,相互吻合。一方面说明我们克隆的家蚕BmPAH基因编码的蛋白具有羟化苯丙氨酸转变成酪氨酸的活力,是名副其实的PAH基因；另一方面说明BmPAH基因参与了家蚕胚胎期黑色素合成,是家蚕胚胎期黑色素合成的关键基因之一。用PAH抑制剂注射普斑蚕基础斑纹的一侧,与另一侧未注射的对称斑纹相比,出现注射处斑纹颜色不同程度的变淡或消失的阳性个体,阳性个体发生率随注射剂量的增加而增加,这说明家蚕幼虫斑纹黑色素合成与BmPAH活性密切相关。用PAH抑制剂培养含有半月纹位置的体壁,表皮斑纹处着色很浅或不着色,而用PAH抑制剂和酪氨酸同时培养体壁,表皮斑纹处有明显的黑色素合成沉积,这不仅进一步说明表皮斑纹黑色素合成与BmPAH活性密切相关,而且说明BmPAH催化形成的酪氨酸是幼虫斑纹黑色素合成的主要来源。综合分析PAH抑制剂注射和培养体壁实验结果,认为BmPAH催化苯丙氨酸形成的酪氨酸是家蚕幼虫斑纹黑色素合成的主要来源,苯丙氨酸是家蚕幼虫斑纹黑色素合成的重要前驱物,BmPAH基因参与了家蚕幼虫斑纹黑色素的合成,是家蚕幼虫斑纹黑色素合成的关键基因之一。纵观家蚕整个生命周期,胚胎期着色和幼虫期斑纹重新着色,黑色素类物质合成最为旺盛。苯丙氨酸是家蚕必需氨基酸,家蚕体内不能自己合成。综合家蚕这些生理特点和前面的实验结果分析认为：BmPAH基因参与了家蚕黑色素合成,是家蚕黑色素合成途径上的关键基因之一；BmPAH催化苯丙氨酸转化成酪氨酸是家蚕黑色素合成途径上的一个重要反应步骤,苯丙氨酸是家蚕黑色素合成最前端、最原始的前驱物；苯丙氨酸→→→→酪氨酸是家蚕黑色素合成途径上的第一个反应步骤。4、BmTRH基因克隆、序列分析及表达模式研究本研究克隆的BmTRH基因cDNA序列长1520bp,其中ORF序列长1509bp。BmTRH基因位于家蚕1号染色体nscaf1690上,包含9个外显子和8个内含子,外显子／内含子边界均符合GT-AG规则；BmTRH基因编码502个氨基酸,预测蛋白的分子量大小约为57 kDa,等电点为5.72。预测显示BmTRH蛋白包含一个ACT结构域和典型的羟化色氨酸的催化结构域,无信号肽序列,属非分泌蛋白。RT-PCR检测结果表明,在家蚕幼虫5龄3天11个组织中,BmTRH基因转录本仅在头和中枢神经表达,其中在中枢神经的表达量比头高。以家蚕个体总cDNA为模板,RT-PCR检测家蚕胚胎、幼虫及变态4个发育阶段39个时间点,仅有一个时间点出现了扩增带。5、BmTRH基因原核表达、western blotting及免疫组化分析本研究构建了序列和插入方向都正确的BmTRH基因原核表达载体——BmTRH/pET-28a(+)重组质粒。在原核中表达的蛋白出现于沉淀,经质谱鉴定确实为BmTRH基因编码的蛋白。用BmTRH基因原核表达蛋白免疫家兔获得的多克隆抗体进行免疫印迹和免疫组化实验。western blotting结果显示,家蚕5龄3天幼虫9个组织总蛋白中没有出现BmTRH蛋白的阳性信号；家蚕5龄3天幼虫组织免疫组化显示,在头部有BmTRH蛋白的阳性信号,在中枢神经组织中目的蛋白的信号较强,在体壁、精巢、卵巢等组织没有检测到目的蛋白的阳性信号。免疫组化结果与RT-PCR结果一致,但与免疫印迹的结果不一致,出现这种现象的原因可能是由于目标蛋白在总蛋白中的含量太低以致免疫印迹检测不到所致。BmTRH基因仅在家蚕神经活动非常丰富的头部组织,和专一进行神经活动的中枢神经组织表达,与TRH是合成神经递质5-HT的限速酶,TRH是5-HT能神经元最好的标志相符。更多还原

【Abstract】 Aromatic amino acid hydroxylase(AAAH) is of important physiological action in aromatic amino acid metabolism. Aromatic amino acid hydroxylase(AAAHs) include phenylalanine hydroxylase(PAH), tyrosine hydroxylase and tryptophan hydroxylase(TRH). Phenylalanine hydroxylases(PAH) exist widely in animals,plants and microorganism, is a key and rate-limiting enzyme in Phe metabolism. PAH catalyzes the hydroxylation of phenylalanine to produce tyrosine, which is the only rate-limiting step in Phe normal catabolism in mammals. Phenylalanine hydroxylase gene is closely related to Phenylketonuria in human and is related to melanin synthesis in the epidermis in nematode. PAH gene is related to skin hardening, eye pigment synthesis and nervous activity in Drosophila and is related to immune response melanism in Aedes too, but it not yet has been reported about the gene of encoding phenylalanine hydroxylase in Lepidoptera insects. Silkworm is as a representative in Lepidoptera insects. It is very important significance to make clear the role and function of BmPAH for exerting adequately model fuction of silkworm to build new method and new technology in controlling Lepidoptera pest in agriculture and forestry. Meanwhile, it is of important reference value for PKU treatment in human.Tryptophan hydroxylase(TRH) is a key and rate-limiting enzyme in serotonin(5-HT) synthesis, its activity determines the content of serotonin, and becomes a good marker for 5-HT synthesis in neurons. Serotonin distributes widespread in nervous system in vertebrates and invertebrates, and participates in many kinds of actions and development modulators.Clinically, polymorphism of tryptophan hydroxylase(TRH) gene is assosiated with many mental disorders, for example depression, obsessive compulsive behavior, anorexia, bulimia, sudden infant death syndrome, Alzheimer’s disease, and schizophrenia and so on. In insects,the reports about tryptophan hydroxylase(TRH) are very poor, but not in Drosophila. Silkworm as a model organism, it is very important significance to clone and express of tryptophan hydroxylase(TRH) gene in silkworm for promote identification on neuronal morphology, type, distribution of TRH gene as well as the connections between neurons of insects.Based the above, the clone, expression and function of phenylalanine hydroxylase(PAH) gene and tryptophan hydroxylase(TRH) in silkworm had been done.The main results are as follows:1 Clone, sequence analysis and expression pattern of BmPAH geneThe cDNA of BmPAH cloned in the study is 1433bp long, its ORF is 1371bp. BmPAH locates on nscaf 2734 of chromosome 1, containing 6 exons and 5 introns, and the border of exons/introns is accord with GT-AG rule; BmPAH encodes 456 amino acids, the size of predicted protein is 52kDa, its PI is5.46. The analysis of protein domain indicated that it contains complete ATC domain and typical phenylalanine domain, no signal peptide sequence. It is belong to a non-secretory protein.The results of tissues expression analysis by RT-PCR showed the transcript of BmPAH expressed highly in testis,ovary, head, skin and fat body of 3-days-old 5th instar larvae of silkworm, expressed not highly in blood, and expressed lowly or no expression in silk gland, midgut and Malpighian tubes.The results of RT-PCR was completely consistent with microarray data.Based on individual total cDNA as template, the experimental results of RT-PCR showed the expression amount of the transcription of BmPAH gene in the silkworm embryo was different from the size of embryonic development, big embryoes developed longer time were higher expression amount than small embryoes developed shorter time. It is consistent with synthetizing largly melanin in the latter embryo development. The transcript of BmPAH expressed in moulting larvae and of every instar. it is consistent with the period which synthetizes largly melanin in larve ecdysis and putting colour on the epidermis again. The amount of expression of BmPAH in total in metamorphosis is obviously lower than two stages, embryo and larvae.2 Prokaryotic expression, western blotting analysis and immunohistochemistry of BmPAH geneThe prokaryotic expression carrier of BmPAH gene--BmPAH/pET-28a(+) recombinant plasmid, was constructed with correct sequence and direction.The protein of prokaryotic expression existed in the deposition, and the protein was identified as the protein encode by BmPAH gene using mass spectrum, and the anti-rabbit antibody.was prepared. The results of western blotting showed that the positive signals were found in fat body, testis and ovary of 3-days-old 5th instar larvae and the fat body had stronger positive signal. There are abundant BmPAH protein in fatbody showed the fatbody of silkworm like human liver which is the main place of phenylalanine metabolism. The results of tissues immunohistochemistry of 3-days-old 5th instar larvae of silkworm showed BmPAH protein existed in tissues of the whole head. This is related to distributing many melanin and growing many sense organs in head. BmPAH protein exist in the dermis and endepidermis of the skin, this is consistent with that the dermis and endepidermis are the places which synthetize melanin in the skin. It expressed higher in the membrane of testis and ovary and expressed in the sperm cell, it maybe relate to genital gianas developing and germ cells maturing. It expressed highly in the center nervous system, maybe relate to nervus actions.The results of immunohistochemistry were concordance with the results of RT-PCR completely, but were inconsistent with the results of western blotting, the reasons for this phenomenon may be that the content of the target protein in total protein was too low so that it was not detected by immunoblotting,3 Study on fuction of BmPAH gene in silkworm synthetizing melaninEgges espawning after three hours were injected using synthetizing dsRNA by oneself or using synthetizing siRNA by company, and some positive embryos which can develop but can not hatch with color lightly or streakily even do not color. The result of real-time PCR showed, the transcript expression of BmPAH gene in these positive embryos were lower than the control’s. It implied that the positive embryos were closely related to the interference of BmPAH gene. The results of the embryos injected siRNA by HPLC-FLU analysis showed that tyrosine leveles in the positive individuals were obviously lower than the controls’and negative individuals’by siRNA, and there were remnants of phenylalanine accumulating in the positive individuals treated by siRNA, guessing that the reasons of BmPAH gene was interfered, expression of BmPAH transcript became low, product of the gene decreased, the action of catalytic reaction became slow and product of tyrosine decreased resulting in the accumulation of phenylalanine. The results of the analysis of HPLC-FLU and Real-time PCR can confirm each other. On the one hand, the results showed the ecoding protein of BmPAH gene cloned by us have activity which can.hydroxylate phe to tyr, it is do real PAH gene. The other hand, the results showed BmPAH gene participated in melanin synthesis and is one of key genes in melanin synthesis in silkworm embryo.Comparing the general dapple on one side of silkworm injected using the inhibitor of PAH with another side symmetrical dapple which don not inject inhibitor, the positive individuals, which dapple turn light or disappear in varying degrees on the injection locus, were found. Incidence of the positive individuals increase with the increasing dose of injection. It indicate that melanin synthesis of silkworm larvae dapple is closely related to activity of BmPAH. Culturing the skins which growes the dapple using the inhibitor of PAH, the dapple color became lightly or disappeared. Culturing the skins using the inhibitor of PAH and tyrosine, there was obviouse melanin synthesis in the dapple of the skin. It not only further indicated that melanin synthesis is closely related to activity of BmPAH, but also indicated tyrosine formed by BmPAH catalyzeing is the main source of epidermis melanin development of larvae maculation. Analysing the results of PAH inhibitor injection and the skin cultureing, we think that tyrosine to which BmPAH catalyzes phe is the main source of melanin synthesis of silkworm larvae dapple. Phe is an important precursor of melanin synthesis in the larvae dapple melanin. BmPAH participates in melanin synthesis and it is one of key genes in the melanin synthesis of larvae dapple melanin.In the whole cyclogeny of silkworm, in the embryonic coloring stage and the larvae dapple newly-coloring, melanin synthesis is the most exuberant. Phe is a necessary amino acid in the silkworm, but it can not synthesize in vivo by silkworm oneself. Based on these physiological properties and the analysis results above, we think: BmPAH participates in melanin synthesis of silkworm and it is one of key genes in the melanin synthesis pathway of silkworm. It is important reaction step that BmPAH catalyze phe to tyr in the melanin synthesis pathway of silkworm. Phe is the frontest and the most primitive precursor; Phe→→→→tyr is the first reaction step in the melanin synthesis pathway of silkworm.4 Clone, sequence analysis and expression pattern of BmTRH geneThe cDNA of BmTRH cloned in the study is 1520 bp, its ORF is 1509 bp. BmTRH locates on nscaf 1690 of chromosome 1, with 9 exons and 8 introns, and the border of exons/introns is accord with GT-AG rule; BmTRH encodes 502 amino acids, the size of the predicted protein is 57kDa, its PI is 5.72. The analysis of protein domain indicated that it contains complete ATC domain and hydroxylateing tryptophan domain and no signal peptide sequence.It is belong to a non secretory protein. RT-PCR in 11 larvae tissues of 3-days-old 5th instar larvae showed that the transcript of BmTRH gene only expressed highly in the head and in the center nervous system and the expression level in center nervous system is higher than that of in head. The results of RT-PCR in 39 time points choosed from four development stages, including embryo, larvae and metamorphosis showed there was an obvious amplification band only in one time point.5 Prokaryotic expression, western blotting and immunohistochemistry of BmTRH geneThe prokaryotic expression carrier of BmTRH/pET-28a(+) recombinant plasmid was constructed with correct sequence and direction. The protein of prokaryotic expression existed in the deposition, and the protein was identified as the protein encode by BmTRH gene using mass spectrum. The results of western blotting showed that no BmTRH positive signal had been testing in 9 tissues of 3-days-old 5th instar silkworm larvae. The resultes of immunohistochemistry of 3-days-old 5th instar silkworm larvae showed BmTRH protein existed in the head and there was the stronger positive signal in the center nervous sestem, no signals were tested in integumentum, testis and ovary. The results of immunohistochemistry were concordance with the results of RT-PCR completely, but were inconsistent with the results of western blotting, the reasons for this phenomenon may be that the content of the target protein in total protein was too low so that it was not detected by immunoblotting. BmTRH only express in the head which nervus act richly and in the center nervous sestem which nervus act specially, and it is coincidence in the condition that TRH is a rate-limiting enzyme in 5-HT synthesis and is the best marker for serotoninergic Neuron.更多还原