荷那龙罗非鱼两种GHSR基因的克隆与序列分析

用RT-PCR和RACE方法,从荷那龙罗非鱼(Oreochromis hornorum)垂体中克隆到生长激素促分泌素受体(GHSR)cDNA全序列。荷那龙罗非鱼GHSR基因具有GHSR-1a与GHSR-1b两个高度保守cDNA序列。GHSR-1a序列全长1 646 bp,包括225 bp的5′非编码区,266 bp的3′非编码区和1 155 bp的开放阅读框,编码384个氨基酸残基,具有7个跨膜结构域结构(transmembrane domains,TM);GHSR-1b序列全长1 877 bp,包括225 bp的5′非编码区,755 bp的3′非编码区和897 bp的开放阅读框,编码298个氨基酸残基,只具有前5个TM,在第6个TM的第4个氨基酸处开始缺失。将GHSR cDNA序列与基因组序列比较发现,这两种cDNA转录本来自同一个基因的不同变体。     

溶藻弧菌acfA基因克隆与生物信息学分析

溶藻弧菌引起海水养殖产业弧菌病的主要病原,附着定植因子基因acfA是溶藻弧菌重要毒力基因之一,其表达产物是溶藻弧菌有效定植在宿主肠道上所必需的蛋白。根据弧菌属细菌acfA基因基因保守区设计引物,采用Touchdown PCR扩增acfA基因部分序列,Inverse PCR和Nested PCR扩增已知序列侧翼序列,成功克隆了溶藻弧菌acfA基因全长。克隆的acfA基因序列全长为743 bp,开放阅读框长度为648 bp组成,共编码215个氨基酸,在5′端上游未发现-35区和-10区序列。演绎的ACFA蛋白N端有18个氨基酸信号肽序列,表明该蛋白是分泌型蛋白;蛋白结构分析表明主要由α螺旋和不规则卷曲构成。BLAST分析表明,该蛋白氨基酸序列与其他弧菌相应蛋白同源性较高,是较保守的外膜蛋白。     

马氏珠母贝接头蛋白CIKS的基因克隆与组织表达分析

【目的】探究马氏珠母贝接头蛋白CIKS(PmCIKS)在马氏珠母贝免疫反应中的作用。【方法】采用cDNA末端快速扩增(RACE)技术获得PmCIKS基因cDNA全长序列,运用生物信息学手段分析该序列,用实时荧光定量PCR(RT-PCR)技术检测PmCIKS基因在马氏珠母贝7个组织中的表达模式。【结果】PmCIKS基因cDNA全长为1 987 bp,其中5′UTR长为228 bp,3′UTR长为148 bp,包含24 bp的ploy A,开放阅读框(ORF)为1 611 bp,编码536个氨基酸,预测其分子质量约为61.3 ku,等电点为7.01。物种间CIKS有较高的保守性。PmCIKS在马氏珠母贝肝胰腺、性腺、闭壳肌、鳃、血细胞、外套膜和足中均有表达,其中在血细胞中表达量最高。     

无乳链球菌sodA基因的克隆、序列分析及原核表达载体构建

无乳链球菌(Streptococcus agalactiae)是引起我国南方罗非鱼链球菌病的主要病原之一,SodA是无乳链球菌重要的毒力因子之一。根据链球菌sod A基因保守区设计引物,采用PCR扩增sod A基因部分序列,反向PCR和巢式PCR扩增其侧翼序列,成功获得无乳链球菌sod A基因。无乳链球菌sod A基因序列全长为699 bp,含开放阅读框609 bp,可编码202个氨基酸,演绎的SodA蛋白主要由α螺旋和不规则卷曲构成。BLAST分析表明,该蛋白氨基酸序列与犬链球菌(S.canis)及副乳房链球菌(S.parauberis)相应蛋白的氨基酸序列相似性分别高达80%和79%。将sodA基因克隆至原核表达载体pET28a上成功构建了重组质粒p ET28a-sod A,导入Escherichia coli BL21(DE3),诱导表达的重组蛋白质分子质量约为24 ku,主要以包涵体形式存在于表达菌中。     

合浦珠母贝丝氨酸蛋白酶抑制因子基因pfser1克隆与表达

【目的】克隆合浦珠母贝(Pinctada fucata)丝氨酸蛋白酶抑制因子pfser1基因,探讨该基因的组织表达及其在天然免疫过程中的作用,以及与生物矿化过程的关系。【方法】通过RACE技术获得pfser1基因的全长,通过生物信息学分析其序列结构特征,利用实时荧光定量PCR方法检测pfser1基因在不同组织中的表达,检测健康合浦珠母贝在被大肠杆菌(Escherichia coli)MG1655刺激后和在贝壳损伤修复实验中pfser1基因表达量的变化。【结果】合浦珠母贝pfser1基因cDNA全长为1240 bp,包含1035 bp的开放阅读框(ORF),编码344个氨基酸,氨基酸序列的功能结构域含有丝氨酸蛋白酶抑制因子Serpin家族保守结构域。pfser1基因在合浦珠母贝各个组织中均有表达,在外套膜边缘膜中表达量最高;大肠杆菌MG1655刺激后,该基因表达量显著升高;在贝壳损伤修复过程中,pfser1基因表达先升高后受到抑制。【结论】pfser1基因所表达的蛋白参与了合浦珠母贝的天然免疫应答过程,并与生物矿化过程有一定关系。     

泰国斗鱼抗缪勒氏管激素(AMH)基因cDNA克隆及表达

利用RACE(Rapid-amplification of cDNA ends)克隆技术从泰国斗鱼(Betta splendens)精巢中克隆抗缪勒氏管激素(Anti-Müllerian hormone,AMH)基因的c DNA序列,分析该基因序列与其他物种的差异,并用RT-PCR半定量方法分析其组织表达的性别差异。结果表明:泰国斗鱼AMH基因的cDNA序列全长为1 804 bp,其中开放读码框为1 596 bp,编码532个氨基酸残基,与其他物种的氨基酸序列差异较大,同源性最高仅55%;AMH前体蛋白的系统进化树显示,泰国斗鱼与尖吻鲈(Lates calcarifer)亲缘关系最近;AMH基因的组织表达有明显的组织特异性及性别差异,在性腺中表达量最高,雄性个体脑、脾和肌肉次之,垂体、肝、肾、心和肠中未检测到AMH表达,雌性个体脾、心脏和肌肉次之,脑、垂、肝和肾中未检测AMH表达。     

红笛鲷IRAK-1基因克隆及哈维弧菌感染后的组织表达

用同源克隆和RACE的方法克隆红笛鲷(Lutjanus sanguineus)白细胞介素1受体相关激酶1(Interleukin-1receptor-associated kinase 1,IRAK-1)基因的cDNA全序列,并用荧光定量PCR分析其在健康鱼及哈维弧菌感染后红笛鲷的组织表达。结果表明,该序列全长3 207 bp(登录号KF728204),包含5′非编码区(UTR)202 bp,3′UTR752 bp,开放阅读框(ORF)2 253 bp,编码750个氨基酸。根据推导的氨基酸序列预测其蛋白分子质量为82.6 ku,理论等电点为6.59。IRAK-1包含1个N端死亡结构域、proST结构域、中央激酶结构域和C末端结构域。荧光定量PCR分析显示,红笛鲷IRKA-1基因在肝脏和皮肤的表达量最高,其次是心脏、鳃、肌肉和胸腺,其余组织的表达量较低。哈维弧菌侵染红笛鲷后,各组织中IRAK-1基因mRNA表达量均呈上调趋势,肝组织中变化最显著。     

大刺鳅mstn基因克隆及表达分析

【目的】了解大刺鳅(Mastacembelus armatus)肌肉生长抑制素(myostatin)基因mstn的序列及其在生长发育中的调控功能。【方法】利用RACE方法克隆大刺鳅mstn全长cDNA序列,采用荧光定量PCR分析其在不同组织和胚胎发育阶段的表达情况。【结果与结论】大刺鳅mstn cDNA序列全长2 690 bp,包含200 bp 5′非编码区、1 359 bp 3′非编码区和1 131 bp开放阅读框,编码376个氨基酸。前22个氨基酸残基为信号肽,第37～254位氨基酸残基为TGF-β前肽域,第282～376位是TGF-β结构域,具蛋白酶水解位点RARR和C端生物活性区的9个保守半胱氨酸残基。系统发育分析表明,大刺鳅MSTN氨基酸序列与合鳃目、鲈形目鱼类同源性较高,与哺乳动物、鸟类、爬行类、两栖类同源性较低。实时定量PCR分析表明,大刺鳅mstn基因在各组织中均有表达,肌肉中表达量最高,眼、脑次之,鳃表达量最低;大刺鳅胚胎发育的各阶段mstn基因均有表达,囊胚期表达量最高,其次为多细胞期,出膜期表达量最低。     

勒氏笛鲷(Lutjanus russellii)肌肉生长抑制素基因的克隆与分析

以勒氏笛鲷(Lutjanus russellii)基因组DNA为模板,采用同源克隆的方法,获得2 887 bp的肌肉生长抑制素(myostatin,MSTN)基因组序列,该MSTN序列具有3个外显子和2个内含子,包括101 bp的5′-UTR、385bp的外显子1、354 bp的内含子1、370 bp的外显子2、761 bp的内含子2、381 bp的外显子3和1 932 bp的3′-UTR。整个开放阅读框编码了378个氨基酸,前面的22个氨基酸为信号肽,具有9个保守的半胱氨基酸及一个RVRR的蛋白酶解加工位点。氨基酸序列分析发现,该基因编码的蛋白质与其他鱼类的I型同源性较高,与其他鱼类的2型MSTN同源性较低,且与鲈形目的同源性最高,与鲤形目的同源性较低,与人、鼠和鸡的同源性最低。采用邻接法(Neighbor-Joining)构建的MSTN的系统发育树表明,勒氏笛鲷MSTN与鲈形目鱼类的狼鲈属亲缘关系较近,且与鱼类的MSTN-1聚为1支。这表明该基因属于Ⅰ型MSTN基因。     

一株H_5N_1亚型禽流感病毒株NA基因的克隆与序列分析

用RT-PCR方法从1个H5N1亚型禽流感病毒分离株A/Chicken/Guangdong/DH/1997扩增NA基因cDNA片段,将其克隆至pMD18-T载体,获得重组质粒pMD-NA,并对其核苷酸序列进行测定和分析。结果表明,该毒株的NA基因长度为1350bp,编码449个氨基酸,与其它H5N1亚型AIV分离株的核苷酸序列同源性为97.0%～99.4%,氨基酸序列同源性为97.7%～99.1%,提示禽流感病毒NA基因保守性较高。NA基因氨基酸序列的聚类分析表明该毒株与来自香港的A/Pheasant/HK/FY155/01和A/Ch/HK/FY150/01两个分离株处于同一进化枝,亲缘关系较近。     

Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXXATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.     

两个奥利亚罗非鱼群体热休克蛋白70基因序列比对分析

采用RACE技术,对两个奥利亚罗非鱼群体(Oreochromis aurea)(美国奥利亚和中国奥利亚)热休克蛋白Hsp70基因完整编码区(code sequences,CDS)cDNA的进行克隆测序。序列分析结果表明:两个奥利亚罗非鱼群体热休克蛋白Hsp70基因CDS序列完全相同,全长1 923 bp,编码640个氨基酸,相对分子质量为70.29×103,理论等电点5.462,均具有Hsp70家族的3个签名序列:IDLGTTYS、IFDLGGGTFD、VVLVGGSTRIPKIQK;核定位信号标签KRKHKKDISQNKRALRR;Dank特征基序DLGTT-S-V;胞质Hsp70特征基序EEVD;靠近C端的GGMP4肽序列;2个糖基化位点NKSI和NVSA。对所得基因序列与已发表的青锵(Oryzias latipes)等物种Hsp70基因的氨基酸序列进行同源性比较,发现两个奥利亚罗非鱼群体与莫桑比克罗非鱼最高99.4%,与牙鲆最低83.9%;系统进化树分析表明奥利亚罗非鱼的Hsp70 cDNA序列与青鳉等物种的Hsp70基因聚在一支,而与牙鲆的Hsp70基因相分离。     

cDNA cloning and expression of a collectin from red-spotted grouper （Epinephelus akaara）

Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens.We obtained the complete cDNA of a C-type lectin(EALec1) from Epinephelus akaara using RACE.The complete EALec1 cDNA sequence was 827 bp.The 5-UTR and 3-UTR were 28 bp and 151 bp,respectively,in length.The sequence also contained a polyadenylation signal AATAAA and a poly(A) tail.The EALec1 cDNA encodes polypeptides with 215 amino acids,including a signal peptide of 31 amino acids.Th...     

红笛鲷α-珠蛋白和β-珠蛋白基因的克隆及序列分析

应用已知的标签序列通过RACE-PCR和RT-PCR方法成功克隆红笛鲷(Lutjanus sanguineus)α-珠蛋白和β-珠蛋白基因的全长cDNA序列。α-珠蛋白和β-珠蛋白基因的cDNA全长分别为560和563 bp,开放阅读框分别为429和444 bp,各编码143和148个氨基酸,理论相对分子质量分别为15690和16400,理论等电点为7.79和6.61。氨基酸序列BLAST结果表明,红笛鲷α-珠蛋白基因和β-珠蛋白基因编码的氨基酸序列与其他鱼类的相似性分别在59%~79%和55%~80%之间。用邻接法(Neighbor-Joining)构建的α-珠蛋白基因和β-珠蛋白基因的系统发育树表明,红笛鲷与真鲷(Pagrus major)、金头鲷(Sparus aurata)和鰤(Seriola quinqueradiata)等亲缘关系较近。     

Cloning and sequence analysis of a full-length cDNA of <Emphasis Type="Italic">SmPP1cb</Emphasis> encoding turbot protein phosphatase 1 beta catalytic subunit

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 （PP1） is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase l（PPlcb）, was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPPlcb, by the rapid amplification of cDNA ends （RACE） technique. The cDNA sequence of SmPPlcb we obtained contains a 984 bp open reading frame （ORF）, flanked by a complete 39 bp 5＇ untranslated region and 462 bp 3＇ untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B （PP2B）. And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPPlcb is extremely conserved in both amino acid and nucleotide acid levels compared with the PPlcb of other vertebrates and invertebrates, and its Kozak motif contained in the 5＇UTR around ATG start codon is GXXAXXGXXATGG, which is different from mammalian in two positions A6 and G3, indicating the possibility of different initiation of translation in turbot, and also the 3＇UTR of SmPPlcb is highly diverse in the sequence similarity and length compared with other animals, especially zebraf＇lsh. The cloning and sequencing of SmPPlcb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.     

Characterization of SNAP-25 gene from marine teleostean, Lateolabrax japonicus

The t-SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) plays an essential role in regulating fusion between the vesicle and plasma membranes during exocytosis. To clone and characterize SNAP-25 gene, the first step in the functional study of SNARE proteins in marine teleostean, was to obtain the cDNA of sea perch SNAP-25 (SPsn25) by RT-PCR and RACE-PCR amplification of a Japanese sea perch. The full-length cDNA of 831bp contains a CDS of 615 bp, coding 204 amino acid residues, and a 5′UTR of 219bp. Bioinformatic analysis revealed that SPsn25 corresponds with SNAP-25a isoform and shares 91.1% identity with SNAP-25a of a goldfish and a zebrafish. The SPsn25 expression in both mRNA and protein levels in the Japanese sea perch had been identified through semi-quantitative RT-PCR and Western Blot assay. Together, these data again confirmed the nerve tissue specificity of the fish SNAP-25 gene expression.     

马氏珠母贝热休克蛋白HSP60基因的克隆与表达分析

运用cDNA末端快速扩增（RACE）技术,克隆马氏珠母贝（Pinctadamαrtensii）热休克蛋白HSP60基因 cDNA全序列。序列全长2495坤,开放阅读框（ORF） 1734坤,编码577个氨基酸,预测的分子量约为61.79阳, 理论等电点为5.4905＇非翻译区（ 5＇UTR）长150坤, 3＇非翻译区（3＇UTR）长611 bp。同源性比对分析结果,马 氏珠母贝HSP60基因与与太平洋长牡妨（Crωsos仰a gIgω）和光滑双蹄螺（Biomphalaria glabrata）的同源性较 高,达到75%。氨基酸序列分析显示,马氏珠母贝HSP60氨基酸序列具有典型的rnt-HSP60特征序列、C-末端典型的GGM重复基序和一个ATP结合结构域。荧光定量数据分析发现,该基因在闭壳肌、外套腹、血淋巴、肝膜腺、性腺、躁、等6个组织中具有表达,在肝膜腺中表达量最高,腮中次之,而在血液和闭壳肌中仅有少量表达;在脂多糖剌激后,该基因表达水平上调,12 h后达到最大值,之后又逐渐下调,均高于对照组,差异具统计学意 义（P〈0.05）。