罗氏沼虾Rab11基因cDNA克隆及其组织表达分析

为了发掘罗氏沼虾(Macrobrachium rosenbergii)免疫相关基因, 研究Rab蛋白(Ras-related proteins inbrain)在罗氏沼虾免疫应答中发挥的作用, 研究采用RACE-PCR技术克隆了罗氏沼虾Rab11基因全长cDNA序列, 记为MrRab11。全长1381 bp, 包括226 bp的5'UTR, 511 bp的3'UTR和645 bp的开放阅读框, 编码214个氨基酸, 含有一个Rab结构域。氨基酸序列比对显示, 罗氏沼虾与冈比亚按蚊(Anopheles gambiae)、蚤状溞(Daphniapulex)、叶蝉(Homalodisca vitripennis) Rab11一致性分别为82%、83%和82%。软件预测, MrRab11编码的蛋白分子量约为23.75 kD, 等电点约为5.34。实时荧光定量表达分析表明, MrRab11基因在罗氏沼虾各组织中都有表达, 肝胰腺中的表达量最高, 其次是肌肉和肠道。在阴沟肠杆菌(Enterobacter cloacae)感染12h后, 罗氏沼虾肝胰腺中MrRab11的表达量上升, 显著高于对照组(P0.05), 推测这是MrRab11对阴沟肠杆菌的应激表达,MrRab11在肝胰腺中参与了罗氏沼虾免疫应答过程。     

罗氏沼虾MrLc3a基因cDNA的克隆及在副溶血弧菌胁迫下的表达分析

虾类和果蝇同属节肢动物.果蝇的相关研究表明自噬与免疫关系密切,而虾类自噬机制研究鲜少.微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain 3,Lc3)与自噬基因Atg8同源,其与自噬体的形成密切相关,是自噬活性的标志分子.本研究利用RACE技术克隆了罗氏沼虾的MrLc3a基因的全长cDNA,用RT-qPCR检测了该基因在罗氏沼虾主要组织中的表达量;并研究了正常和副溶血弧菌感染两种情况下MrLc3a基因和免疫基因Relish的表达变化情况,为其在病害防御方面的应用提供了前期数据.试验结果表明:MrLc3a基因全长653 bp,其中包括195 bp的5'-UTR、378 bp的ORF开放阅读框和80 bp的3'-UTR,共编码126个氨基酸;序列比对结果显示,其编码的氨基酸序列和南美白对虾Lc3a编码的氨基酸序列具有较高的同源性,并在系统发育树上聚为一支;RT-qPCR结果显示,MrLc3a基因在罗氏沼虾各个组织均有表达,其中在脑、鳃、胃中的表达量较高,在肝胰腺和性腺中的表达量较少;副溶血弧菌感染罗氏沼虾后显著影响了MrLc3a和Relish基因在罗氏沼虾肝胰腺组织中的转录情况,MrLc3a和Relish基因随时间变化都呈现出先上升后下降的趋势,表明MrLc3a基因通过参与细胞自噬过程而参与了免疫反应.     

罗氏沼虾蜕皮激素受体EcR基因的克隆及表达分析

为获得罗氏沼虾蜕皮激素受体(ecdysone receptor, EcR)基因编码序列,并检测mRNA表达模式。本实验拟利用克隆测序技术,以肝胰腺为实验素材,扩增EcR基因的编码区;借助生物信息学手段对所得序列进行分析;采用实时荧光定量PCR技术检测EcR基因在肌肉和肝胰腺等8个组织中的m RNA表达量。结果表明,罗氏沼虾EcR基因的编码区长度为1 716 bp,共编码571个氨基酸。物种间同源性比较分析发现,罗氏沼虾EcR基因序列与褐虾、黑背陆地蟹、日本沼虾、美洲龙螯虾、大西洋砂招潮蟹、蓝蟹、三疣梭子蟹、中华绒螯蟹和拟穴青蟹的同源性分别为90.7%、84.1%、83%、81%、81%、80.6%、79.8%、77.2%和75.9%。组织表达谱结果显示,EcR基因在所检8种组织中广泛表达,且不同性别相同组织间和相同性别不同组织间EcR的表达均存在不同程度的差异性。其中EcR基因在雄虾的鳃组织中表达量最高,在肌肉组织表达量最低;在雌虾的卵巢组织中高表达,在腹节神经组织中低表达。此外,EcR基因在雌虾的性腺、肠和肝胰腺组织中的表达量极显著或显著高于雄虾(p0.01或p0.05),在鳃组织中极显著低于雄虾(p0.01)。本研究成功克隆了罗氏沼虾的EcR基因,检测到EcR基因在各组织中广泛表达,且在不同性别相同组织和相同性别不同组织中存在显著差异。本实验为进一步研究EcR基因与生长发育调控作用提供了理论依据。     

罗氏沼虾蜕皮周期中内分泌调控和蜕皮信号通路相关基因的表达分析

蜕皮是罗氏沼虾重要的生理过程,为了探究罗氏沼虾蜕皮周期中内分泌调控与蜕皮通路中相关基因在蜕皮周期中的表达模式,阐明罗氏沼虾蜕皮的分子调节通路。本研究测定了罗氏沼虾肝胰腺和血淋巴组织中蜕皮周期内蜕皮相关酶活性（谷氨酰胺合成酶,β-N乙酰氨基葡萄糖苷酶和几丁质酶）与蜕皮激素含量,并通过RT-qPCR分析了蜕皮信号通路中Mr-ETHR、MrFTZ-F1以及RXR、ECR和MIH基因在罗氏沼虾不同蜕皮周期内的表达模式。酶活测定结果表明,谷氨酰胺合成酶在血淋巴组织中活力高于肝胰腺组织（P<0.05）;β-N乙酰氨基葡萄糖苷酶在肝胰腺和血淋巴组织中蜕皮前期的活力远高于蜕皮后期（P<0.05）。在肝胰腺中几丁质酶在蜕皮后期活力显著高于其他时期（P<0.05）。肝胰腺和血淋巴组织中蜕皮激素含量在蜕皮间期最低,蜕皮后期达到最高,呈上升趋势。通过PCR扩增与测序验证获得了Mr-ETHR、Mr-FTZ-F1基因ORF全长序列,Mr-ETHR基因ORF全长1 173 bp,编码390个氨基酸;Mr-FTZ-F1基因ORF全长为1 206 bp,编码401个氨基酸。荧光定量结果表明Mr-ETH...     

罗氏沼虾幼体精氨酸激酶基因的cDNA克隆及其在双顺反子病毒感染后的表达特征

为探求罗氏沼虾精氨酸激酶（Macrobrechium rosenbergii arginine kinase,MrAK）基因特征和与病毒感染的相关性,研究通过AK基因mRNA全长克隆,并采用QPCR检测病毒感染前后不同时间点罗氏沼虾幼体AK基因的转录差异。最终克隆得到的AK序列全长为1740 bp（GenBank:KT970484）,其开放阅读框包含1068个碱基,编码356个氨基酸;同源性分析显示MrAK基因编码区蛋白与多齿新米虾、南极磷虾、鲑鱼海虱和安氏伪镖水蚤的同源性分别为97%、82%、83%和79%;系统进化树分析表明,该基因与多齿新米虾的AK聚在同一分支簇,而蟹、对虾和螯虾聚在另一分支;氨基酸结构分析表明,其存在一个可能的ATP:胍基磷酸转移酶的活性位点（Cys286-Pro287-Thr288-Asn289-Leu290-Gly291-Thr292）;病毒感染罗氏沼虾幼体后,其AK基因表达在第9h开始出现显著性上调,在12h时达到峰值,随后开始降低。以上研究结果表明,AK基因在无脊椎甲壳动物中存在多样性,其蛋白结构存在保守性,并且其在罗氏沼虾病毒感染过程中起到潜在的能量供给调节作用。     

壬基酚和雌二醇干扰罗氏沼虾卵黄蛋白原VTG基因表达的效应

对刚孵化后的罗氏沼虾(Macrobrachium rosenbergii)亲虾用高低两个浓度的壬基酚(nonylphenol,NP,100μg/L和0·01μg/L)和雌二醇(estradiol,E2,1μg/L和0·01μg/L)进行浸泡处理,分别于3d和5d对罗氏沼虾肝胰腺和卵巢中卵黄蛋白原(vitellogenin,VTG)基因表达变化进行半定量分析。结果显示,NP和E2能够提高罗氏沼虾肝胰腺和卵巢中卵黄蛋白原VTG基因的表达。100μg/LNP对罗氏沼虾表现出雌激素效应,0·01μg/LNP作用效果不明显;而E2则在1μg/L和0·01μg/L两个浓度下均对罗氏沼虾有较强的雌激素效应。在100μg/LNP作用下,卵巢VTG表达量3d和5d均保持较高水平,无明显下降,其他实验组均是3dVTG基因表达量升高,5d后表达量较3d表达量有所降低。结果表明,与其他动物一样,NP对罗氏沼虾具有内分泌干扰作用。     

日本沼虾三群体线粒体16S rRNA基因片段序列的差异与系统进化

通过对日本沼虾(Macrobrachium nipponense)3个群体线粒体DNA 16S rRNA基因片段进行扩增和测定,得到长度为495bp的片段,其碱基A、T、G和C的平均含量分别为28.6%、36.1%、22.7%和12.5%,AT含量明显高于GC含量。通过对日本沼虾16SrRNA基因片段遗传特征的研究发现其种内变异很小,在3个群体中只有5个位点发生转换。另外,利用其454bp的同源序列,以中国明对虾(Fenneropenaeus chinensis)为外群探讨了沼虾属日本沼虾、罗氏沼虾(M.rosenbergii)等8种沼虾的系统进化关系。用MEGA3.1软件中的NJ法构建的分子进化树,日本沼虾3个群体先聚在一起后与海南沼虾聚在一起;另外,罗氏沼虾与马氏沼虾、短腕沼虾与贪食沼虾亲缘关系较近先聚在一起,然后再与大臂沼虾和等齿沼虾聚在一起,最后才与外群中国明对虾聚在一起。     

生长滞缓与正常罗氏沼虾转录组差异分析

旨在利用浙江地区生长正常和生长滞缓两种生长状态下的罗氏沼虾,通过转录组测序技术,比对基因表达量的差异,探究生长滞缓的原因.本研究采用IlluminaHiSeqTM4000测序平台,对存在生长差异的罗氏沼虾的鳃、肝胰腺、肌肉三种组织共18个样本进行了转录组测序分析,将得到的基因片段组装后与NR、GO、KEGG、eggNOG、Swiss-Prot共五大数据库比对注释,然后进行表达差异分析.其中从肝胰腺组织共得到至少35 942 944条Clean read,鳃组织共得到至少34 309 548条Clean read,肌肉组织得到至少34 695 288条Clean read,拼接得到243 414条.通过对三种组织生长正常和生长滞缓样本文库分别进行两两对比,本研究在肝胰腺中发现2 323个基因表达呈显著差异(P＜0.05),肌肉中1 505个基因表达呈显著差异(P＜0.05),鳃组织中1 896个基因表达呈显著差异(P＜0.05).三种组织中得到存在共同差异的基因27个,其中24个基因在三种组织中表达趋势一致,其中14个基因被成功注释,新发现10个未被注释的基因,可能与罗氏沼虾生长密切相关的基因.本研究为进一步探究罗氏沼虾生长缓慢原因提供了基础信息.     

斑节对虾溶菌酶基因克隆及序列分析

参考对虾溶菌酶基因和类溶菌酶基因及其他多种生物的溶菌酶基因序列 ,设计并合成引物。运用RT PCR技术 ,从斑节对虾血细胞总RNA中扩增获得特异性片段。所获片段回收纯化后克隆到pGEM TEasyVector系统的T载体上。重组子的序列分析表明 ,所克隆的斑节对虾溶菌酶基因片段长 6 5 8bp ,包括溶菌酶基因开放阅读框 (ORF)4 77bp和 3′端非编码区的 181bp。 4 77bpORF共编码 15 8个氨基酸 ,包括溶菌酶成熟肽 14 0个氨基酸残基和信号肽 18个氨基酸残基。斑节对虾溶菌酶成熟肽推测分子量为 16 ,32kd ,等电点为 8 78。与南美白对虾溶菌酶基因的碱基序列及推测氨基酸序列比较 ,同源性分别为 89 5 %和 93 0 % ;与日本对虾类溶菌酶基因的同源性分别为 84 0 %和91 0 %。进一步的序列分析表明 ,斑节对虾溶菌酶氨基酸序列与多种类群生物的c 型溶菌酶氨基酸序列具有较高的同源性 ,并具有与c 型溶菌酶相同的活性位点氨基酸残基Glu51和Asp68,且与活性位点相邻的序列高度保守。斑节对虾溶菌酶氨基酸序列还具有与c 型溶菌酶相同的结构氨基酸——— 8个半胱氨酸残基。因而可认为所克隆的斑节对虾溶菌酶基因属c 型溶菌酶基因。     

日本沼虾含硒谷胱甘肽过氧化物酶全长克隆及表达分析

为探讨日本沼虾(Macrobrachium nipponense)的免疫机制及含硒谷胱甘肽过氧化物酶(Se-GPx)在甲壳动物解毒和免疫应激反应中的作用,本文采用RACE法克隆了日本沼虾Se-GPx cDNA全长,其cDNA全长为908 bp,5′-UTR的长度为91 bp,3′-UTR长为256 bp,包括1个保守的硒代半胱氨酸插入序列(SECIS)和1个polyA尾,开放阅读框长度为561 bp,编码由186个氨基酸组成的多肽,其中,第39个氨基酸为TGA编码的硒代半胱氨酸。同源性和相似性分析显示日本沼虾的Se-GPx在甲壳动物中与罗氏沼虾相似度最高,在脊椎动物中与GPx1和GPx2家族的相似度比较高。日本沼虾Se-GPx基因在血细胞、肝胰腺、肌肉、卵巢、表皮以及大颚器官中都有表达,尤其在血细胞中表达量相对较高。用嗜水气单胞菌(Aeromonas hydrophila)刺激后3h和6h,血细胞Se-GPx基因表达量显著升高。结果表明,日本沼虾Se-GPx作为一种重要的抗氧化酶,在维护组织正常功能方面及免疫应激反应中发挥重要作用。     

Cloning and characterization of the tiger shrimp lysozyme

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. A lysozyme gene isolated from tiger shrimp, Penaeus monodon, was cloned, sequenced and characterized. The cDNA consists of a signal peptide of 18 amino acids and a mature peptide of 140 amino acids. The lysozyme is presumed to be a chicken-type lysozyme for it possesses two catalytic sites and eight cysteine residues which are highly conserved across species of chicken-type lysozymes. The lysozyme cDNAs of Penaeus semisulcatus, Litopenaeus vannamei, Macrobrachium nipponense and Macrobrachium rosenbergii were also cloned. High similarities existed among shrimp and prawn lysozymes but phylogenetic relationship of shrimps and prawns based on lysozyme molecules did not quite consistent with traditional taxonomic classification. High mRNA expression was detected in hepatopancreas, haemocytes and gill of tiger shrimp. Recombinant lysozyme exhibited potent lytic activities against fish pathogens providing evidence of the involvement of lysozyme in shrimp immunity.     

An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity

Lysozymes function in animal immunity. Three types of lysozyme have been identified in animal kingdom and most lysozymes identified from bivalve molluscs belong to the invertebrate (i) type. In this research, we cloned and sequenced a new i-type lysozyme, named MmeLys, from the Asiatic hard clam Meretrix meretrix. MmeLys cDNA was constituted of 552 bp, with a 441 bp open reading frame encoding a 146 amino acid polypeptide. The encoded polypeptide was predicted to have a 15 amino acid signal peptide, and a 131 amino acid mature protein with a theoretical mass of 14601.44 Da and an isoelectric point (pI) of 7.14. MmeLys amino acid sequence bore 64% identity with the Manila clam (Venerupis philippinarum) i-type lysozyme and was grouped with other veneroid i-type lysozymes in a bivalve lysozyme phylogenetic tree predicted using Neighbor-Jointing method. Recombinantly expressed MmeLys showed lysozyme activity and strong antibacterial activity against Gram positive and Gram negative bacteria. MmeLys mRNA and protein were detected to be mainly produced in hepatopancreas and gill by the methods of semi-quantitative RT-PCR and western blotting. In addition, MmeLys gene expression increased following Vibrio parahaemolyticus challenge. Results of this research indicated that MmeLys represents a new i-type lysozyme that likely functions in M. meretrix immunity.     

Isolation and characterization of a cDNA encoding for a lysozyme from the gut of the reduviid bug Triatoma infestans

We have isolated and characterised a Triatoma infestans cDNA encoding a lysozyme. A 174-bp fragment was amplified by PCR using degenerate oligodeoxyribonucleotide primers derived from the known amino acid sequences of lysozyme from other insects. This PCR fragment was used to screen a cDNA gut library of T. infestans. A clone containing the 3'-end of the lysozyme cDNA (219 bp) was isolated and sequenced. RACE was used to amplify the 5'-end of the lysozyme cDNA. After sequencing the complete lysozyme cDNA, the deduced 417 amino acid sequence showed high identity (40-50%) with other chicken-type lysozymes. The amino acid residues responsible for the catalytic activity and the binding of the substrate were essentially conserved. The expression pattern of the lysozyme gene in bugs at different molting and feeding states showed that this gene was upregulated in the digestive tract directly after the molt and after feeding. Additionally, this lysozyme gene was expressed differently in the different regions of the digestive tract, strongly in the cardia and stomach, the anterior regions of the midgut, and only traces of lysozyme mRNA could be detected in the small intestine, the posterior region of the midgut.     

Hepatopancreas is the likely organ of vitellogenin synthesis in the freshwater prawn, Macrobrachium rosenbergii

The objective of the present study was to investigate the source of vitellogenin in the freshwater prawn, Macrobrachium rosenbergii. Ovarian development of M. rosenbergii was classified into five stages (stage I-V). Vitellin/vitellogenin was detected in the ovary and the hepatopancreas in different stages by native-PAGE and Western blotting. Two and three subunits of vitellin were observed in the ovary at the early- (I-II), mid- and late- (III-V) stages, respectively. The subunit of vitellogenin was not detected in the hepatopancreas at different stages of prawns. Hepatopancreas had positive immunocytological staining (against vitellin antibody) in different ovarian stages of prawn. Only vitellogenic oocyte but not previtellogenic oocytes and follicle cells had a positive immunocytological staining. Hepatopancreas could synthesize radiolabeled immunoreactive proteins after incubation with radiolabeled glycine on the basis of immunoprecipitation (against vitellin antiserum). Therefore, it is concluded that hepatopancreas is the most likely organ to synthesize vitellogenin in the freshwater prawn, M. rosenbergii.     

Identification and expression analysis of three c-type lysozymes in Oreochromis aureus

Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. Three genes encoding chicken-type (c-type) lysozymes, C1-, C2-, C3-type, were obtained from tilapia Oreochromis aureus by RT-PCR and the RACE method. Catalytic and other conserved structure residues required for functionality were identified. The amino acid sequence identities between C1- and C2-type, C1- and C3-type, C2- and C3-type were 67.8%, 65.7% and 63.9%, respectively. Phylogenetic tree analyze indicated the three genes were firstly grouped to those of higher teleosteans, Pleuronectiformes and Tetraodontiformes fishes, and then clustered to those of lower teleosteans, Cypriniformes fishes. Bioinformatic analysis of mature peptide showed that the three genes possess typical sequence characteristics, secondary and tertiary structure of c-type lysozymes. The three tilapia c-type lysozymes mRNAs were mainly expressed in liver and muscle, and C1-type lysozyme also highly expressed in intestine. C1-type lysozyme mRNA was weakly expressed in stomach, C2- and C3-type mRNAs were weakly expressed in intestine. After bacterial challenge, up-regulation was obvious in kidney and spleen for C1-type lysozyme mRNA, while for C2- and C3-type lysozyme obvious increase were observed in stomach and liver, suggesting that C1-type lysozyme may mainly play roles in defense, while C2- and C3-type lysozyme mainly conduct digestive function against bacteria infection. All the three c-type recombinant lysozymes displayed lytic activity against Gram-negative and Gram-positive bacteria. These results indicated that three c-type lysozymes play important roles in the defense of O. aureus against bacteria infections.