海带雄配子体抑制消减cDNA文库的生物信息学分析

对海带(Landnaria japonica Axesch.)雄配子体抑制消减cDNA文库进行生物信息学分析,并通过Northern印迹杂交进一步验证了雄配子体2个优先表达的基因.结果表明,(1)由627个克隆产生的618条高质量cDNA可聚类成187条非冗余序列(NRS);其中93条Contig由524条cDNA拼接而成,剩下的94条为Singleton(只有单条cDNA),冗余率达69.74%.(2)5条NRS因与rRNA基因相匹配而不进行分析;60条NRS与GenBank中注释蛋白基因显著匹配;122条NRS无显著匹配.(3)大部分已知功能的基因与物质和能量代谢(26.7%)、生长发育(13.3%)有关,其中与叶绿体光捕获蛋白复合体有关的基因有106条cDNA,所涉及的有lhcf6基因(62条cDNA)与fcp基因(12条cDNA)等.(4)经Northern印迹杂交证实lhcf6和fcp2个基因在处于营养生长期的雄配子体中优先表达.(5)90条Contig的总G+C含量平均值为52.58%,表明了密码子的使用对G、C有明显的倾向性;20个推测功能蛋白(1520密码子)密码子第三位碱基的使用偏好C(40.7%),其次是G(33.9%);终止密码子的使用偏好TGA(54.5%).本研究为海带雌、雄配子体差异表达基因克隆、分析和功能基因组学等研究提供了信息.     

达氏鳇肌肉组织转录组测序和功能分析

为发掘达氏鳇的功能基因,采用新一代高通量测序技术对达氏鳇肌肉组织转录组进行测序。结果获得原始数据14 447 211 200 bp,拼接获得了55 531条单基因序列(unigene),长度范围300~32 613 bp,平均长度941 bp。利用生物信息学方法对unigene进行了非冗余蛋白质数据库(Nr)相似搜索,此外还进行了GO的功能注释和KEGG代谢通路分析。结果一共有20 735条unigene(37.34%)与Nr数据库中的己知基因同源;根据GO功能可分为生物过程、细胞组分和分子功能3大类56分支;依据KEGG代谢通路分析可以分成290类。     

翘嘴鳜生长激素cDNA克隆及其真核表达载体的构建

根据GenBank上大眼鳜生长激素cDNA序列(收录号:AY155227)设计了1对特异性引物,应用RT-PCR技术从翘嘴鳜(Siniperca chuatsi)脑垂体中克隆了生长激素基因(scGH)编码区序列并对其真核表达载体的构建进行了研究。结果显示:扩增片段全长615 bp,共编码204个氨基酸。该序列与GenBank上大眼鳜生长激素cDNA序列同源性为99.84%。将scGHcDNA序列定向插入pYES2/CT真核表达载体中,经过筛选、酶切和测序,证明重组子中确实插入了翘嘴鳜GH片段。     

鳜胃蛋白酶原基因cDNA全长的克隆与序列分析

利用RT–PCR和cDNA末端快速扩增法(RACE)克隆鳜(Siniperca chuatsi)胃蛋白酶原基因cDNA全长序列,并对该基因的结构特征和系统进化关系进行了分析。鳜胃蛋白酶原基因cDNA序列全长1367 bp,5′端非翻译区43 bp,3′端非翻译区187 bp,开放阅读框(ORF)1137 bp,共编码378个氨基酸。鳜胃蛋白酶原氨基末端存在信号肽和激活肽序列,序列中含有催化活性必需的2个天冬氨酸残基和构成二硫键的6个半胱氨酸残基。鳜胃蛋白酶原氨基酸序列与其他脊椎动物胃蛋白酶原氨基酸序列的同源性为59.9%～91.2%,表明胃蛋白酶原基因在脊椎动物的长期进化中比较保守。鳜胃蛋白酶原基因的成功克隆不仅为进一步研究该基因的时空表达奠定基础,而且为鱼类胃蛋白酶原的分子特征和进化提供了新的资料。     

鳜脂蛋白脂酶和肝脂酶基因结构与组织表达

为了研究鳜(Sinioerca chuatsi)脂蛋白脂酶(LPL)、肝脂酶(HL)基因结构与功能关系,首先采用RT-PCR及RACE法,克隆得到鳜肝脏LPL与HL基因cDNA全序列.鳜肝脏LPL基因cDNA全长为2089 bp,其中开放阅读框(ORF)为1 548 bp,编码515个氨基酸.鳜HL基因cDNA全长为1 964 bp,其中ORF为1 494 bp,编码497个氨基酸.进化树分析显示,鳜LPL与HL基因与其他脊椎动物的LPL、HL基因各自聚集成簇.对鳜基因组进行PCR获得鳜LPL与HL全基因组DNA序列,与其他脊椎动物基因结构相似,鳜LPL基因由10个外显子和9个内含子组成,全长7 392 bp;鳜HL基因南9个外显子和8个内含子组成,全长8 837 bp.应用Genome Walker方法在鳜克隆得到一段长为1 071 bp的LPL和一段长为2 173 bp的HL基因5'侧翼Ⅸ序列,并利用相关软件预测其中具有多个保守的顺式调控元件.组织表达研究显示与易患动脉粥样硬化的人类LPL不同,鳜LPL基因在所有被检测组织中均有表达:在脂肪组织中大量表达,其次是肝脏、脑、肠道、肌肉,在脾中表达量最低;而鳜HL基因的表达则与人类相似,只在肝脏中表达.本研究将为深入探讨机体脂质代谢调节机制以及LPL、HL在防治动脉粥样硬化中的作用奠定良好基础.     

孔石莼在干出胁迫下上调表达基因消减cDNA文库的构建及分析

利用抑制消减杂交技术构建了潮间带绿藻孔石莼(Ulva pertusa)在干出胁迫状态下上调表达基因的消减cDNA文库.获得的阳性克隆经测序得到150条上调表达的EST片段,其中21条为冗余序列(RS),137条非冗余序列(NRS).片段中最长833 bp,最短106 bp,平均长度364 bp.参与比对的EST片段按照...     

织锦巴非蛤不同颜色斧足的转录组

为探索织锦巴非蛤斧足颜色差异的分子机制,以指导高品质织锦巴非蛤的选育工作,实验对织锦巴非蛤橘黄色和浅白色的斧足进行二代和三代转录组测序及差异表达分析。二代测序结果显示,6个样品共产生44.82 Gb clean data,GC含量为35.30%～38.21%,Q30碱基百分比均在94.43%以上。三代测序结果显示,橘黄色斧足组和浅白色斧足组分别得到16 968和21 611条高质量转录本,将其与各数据库进行比对,共获得5 058条有注释信息的全长转录本。以三代全长转录本作为参考序列,将二代数据比对回参考序列,鉴定到57个差异表达转录本。其中,橘黄色斧足组相对于浅白色斧足组有34个差异转录本表达量上调,23个差异转录本表达量下调。使用实时荧光定量PCR (qRT-PCR)对随机挑选的12个差异表达转录本进行验证,其结果与转录组分析结果一致。随后,通过生物信息学分析发现,脂肪酸的生物合成和代谢、肌肉成分、能量代谢等都影响织锦巴非蛤斧足颜色的调控,其中过氧化物酶体增值物激活受体(PPARs)调节着脂肪酸的生物合成和代谢,肌钙蛋白是肌肉的重要组成成分,精氨酸激酶(AK)参与了色素富集过程中的能...     

基于RNA-Seq高通量测序技术的大口黑鲈转录组分析

文章以大口黑鲈(Micropterus salmoides)组织作为研究对象,利用RNA-seq技术进行转录本测序和数据分析,经拼接组装,最终获得35 659条unigenes,序列平均长度738 bp,序列长度中位数(N50)为1 052 bp。另外从长度分布与GC含量等方面对unigenes进行评估,数据显示测序质量好、可信度高。使用6大数据库(KOG、Nr、Pfam、Swiss-Prot、GO和KEGG)注释大口黑鲈转录组unigenes,分别对应有15 832、21 279、14 524、16 973、15 024和11 185条unigenes获得注释。其中,5 617条unigenes在以上所有数据库中同时注释成功,17 253条unigenes至少被一个数据库注释。KEGG分析结果显示,获得注释的11 185条unigenes被划分到267个代谢通路中,参与信号转导通路的unigenes数量最多,共有1 349条(12.06%)。另外还检测到4 030个微卫星(SSR)位点。通过对大口黑鲈转录组测序,获得了大量的转录组信息,为大口黑鲈的功能基因克隆、基因组学、遗传多样性分析、分子标记开发及遗传改良等研究奠定了基础。     

哈维氏弧菌感染的杂色鲍全组织均一化cDNA文库的构建

提取了哈维氏弧菌（Vibrio harveryi）感染的杂色鲍（Haliotis diversicolorReeve）总RNA,采用SMART方法合成双链cDNA,并用双链特异核酸酶进行均一化处理。割取0.5~1.0和1.0~3.0kb的片段分别连接pDNR-LIB并转化大肠杆菌（Esherichia coli）,最终构建2种片段大小的杂色鲍全组织均一化cDNA文库。2文库库容均在2.5×105cfu.mL-1左右,PCR检测阳性重组率为100%。随机挑取200个克隆测序,获得高质量EST序列174条。组装后得到149条Unigenes,冗余率为14.37%。序列注释结果表明,有39条Unigene序列与已知基因高度相似。综上所述,文章所建cDNA文库质量良好,可以满足后续研究工作的需要。     

文蛤肠、外套膜和肝胰脏组织 cDNA 文库构建及其 ESTs 序列分析

利用SMART cDNA文库构建试剂盒构建了文蛤(Meretrix meretrix)肠、外套膜和肝胰脏组织的cDNA文库。经测定原始文库滴度分别为2.10×106、1.70×106和1.60×106,重组率高于95%,肠组织文库插入片段长度均大于1000bp,外套膜和肝胰脏组织文库的插入片段长度大于1kb的占87.5%,文库质量符合标准要求。随机选取文库的3168个克隆进行5′端测序,获得高质量表达序列标签(Expressed Sequence Tags,ESTs)3029个(肠:1005,外套膜:1019,肝胰脏:1005),测序成功率95.58%。经质量控制和拼接得到1796个单基因簇(Unigene),其中306个叠联群(Contigs),1490个单一序列(Singletons)。通过Blastx搜索比对、查询和注释分析,共得到已知基因696个(肠:216,外套膜:235个;肝胰脏:245个),占总数38.75%。在1796个单基因簇(Unigene)发现微卫星序列55条,这些存在微卫星位点的序列占整个ESTs数据库的3.1%。     

脊尾白虾血细胞ESTs的生物信息学与微卫星序列特征分析

对前期测序得到的脊尾白虾血细胞2853条EST序列进行了生物信息学和微卫星序列特征分析。EST序列拼接得到1053条Unigenes,包括329条Contigs和724条Singlets。BLAST分析表明,593(56.3%)条Uingenes与数据库中已知基因具有相似性。KEGG代谢途径分析表明,181条Unigenes映射到120条代谢途径。通过EST-SSR分析,共得到416条微卫星序列,检出率为14.58%。其中,两碱基重复序列374条,占89.90%,AG重复类型最多;三碱基35条,占8.41%,AAT重复类型最多;四碱基7条,占1.68%,AAGT重复类型最多。本研究可为脊尾白虾功能基因资源挖掘及分子标记筛选提供有效数据。     

大菱鲆脾脏cDNA文库构建、EST序列分析与免疫抗病相关基因的筛选

以大菱鲆脾脏为材料,构建cDNA文库,通过对文库克隆的序列测定和初步生物信息学分析,共获得3 656个大菱鲆脾脏表达序列标签（EST）。经与GenBank数据库的序列比对后发现,1 891个EST代表了149个已知基因,其余1 765个EST为未知序列。这149个基因大致可分为6类：9个为细胞结构类（6.0%）;26个为代谢类（17.5%）;45个为细胞防御/免疫类（30.2%）;43个为基因/蛋白质表达类（28.9%）;16个为细胞信号转导/细胞交流类（10.7%）;10个无法明确分类（6.7%）。鉴定的与免疫抗病相关重要基因主要有：CC/CXC趋化因子;主要组织相容性复合体（MHC）class I α;C₃补体;天然杀伤细胞增强因子（NKEF）;胸腺素β-4/β-12;干扰素诱导蛋白Gig1;白介素8受体;STAT 4;热激蛋白70/90等。     

Discovery of host defence genes in the Japanese scallop Mizuhopecten yessoensis Jay by expressed sequence tag analysis of kidney tissue

The Japanese scallop Mizuhopecten yessoensis is one of the most important aquaculture mollusca in Japan and China. In the present study, a high‐quality cDNA library of the Japanese scallop was constructed from the kidney tissue. A total of 2919 expressed sequence tags longer than 100 bp were generated from this library. A cluster of 1440 unique sequences, which consisted of 258 contigs and 1182 singletons, was revealed. Based on blast searches, 882 (61.3%) genes had significant (E‐value <1e–5) matches to known sequences in public databases. Among them, >70 genes were involved in stress response, immunity and apoptosis. These results expanded our knowledge of the genetics and physiology of the Japanese scallop, and provided a useful resource for gene discovery for further research of this species.     

Expression of four muscle proteins at different growth stages of Günther's walking catfish Clarias macrocephalus

The complete cDNA sequences of four contractile muscle genes of walking catfish Clarias macrocephalus were characterized by assembling partial EST sequences from a skeletal muscle cDNA library. The four genes were parvalbumin 4 (PV4) (670 bp), troponin C (TnC) (1065 bp), troponin I (TnI) (843 bp) and myosin light chain 3 (MLC3) (953 bp), leading to deduced amino acid sequences of 109, 160, 176 and 150 residues respectively. During the larval stage, TnC mRNA showed the highest levels of expression with a 1.4‐fold increase from day 1 to day 30 post hatch. At 90 days, the relative expression levels of PV4, TnC and MLC3 were the highest, with similar proportions in the skeletal muscle, corresponding to the highest relative growth rate of walking catfish. Expression of the three calcium‐binding proteins remained high in 6‐month‐old fish, with higher levels of PV4. The different proportions of muscle proteins expressed suggested the significance of their contributions to fish growth and appeared to be correlated with the functional properties of muscle cells, which were observed from changes in the swimming activity of the fish.     

Cloning of kuruma prawn Marsupenaeus japonicus crustin-like peptide cDNA and analysis of its expression

ABSTRACT:   Antimicrobial peptides serve as an important component of the innate immune system of all species by functioning to provide a rapid first line defense against infection. Arthropod antimicrobial peptides have been well described in insects, whereas only a few molecules have been identified in crustaceans. Five variants (types 1–5) of Marsupenaeus japonicus crustin-like peptide cDNA that were obtained from a hemocyte cDNA library and polymerase chain reaction (PCR) amplification are reported here. Marsupenaeus japonicus crustin-like peptide type 1, the predominant type, has a cDNA consisting of 679 nucleotides and an open reading frame consisting of 573 base pairs coding for 191 amino acid residues. Other types contain varying glycine-rich repeats at the N-terminal amino acid sequences. The deduced amino acid sequences of these variants are highly similar to those of Litopenaeus setiferus (80% identity), Litopenaeus vannamei (80% identity) and Carcinus maenas crustins (44% identity). Expression of Marsupenaeus japonicus crustin-like peptide mRNA was detected in hemocytes, but not in the heart, hepatopancreas, gill, fore-gut, mid-gut, muscle, subcuticular epithelium or ovary. The expression level of crustin-like peptide mRNA increased significantly 1, 3 and 7 days post-peptidoglycan feeding as determined by quantitative real-time PCR. These results suggest that crustin-like peptide could have an important role in shrimp defense mechanisms.     

Amino acid sequences of α-skeletal muscle actin isoforms in two species of rattail fish, Coryphaenoides acrolepis and Coryphaenoides cinereus

SUMMARY: The cDNA clones of α-skeletal actins were isolated from the skeletal muscle of two species of rattail fish, Coryphaenoides acrolepis and Coryphaenoides cinereus . The complete nucleotide sequences of the cDNA and their deduced amino acid sequences were determined. Each of the two species had two α-skeletal actin cDNA. The nucleotide sequences of the coding region of the two α-skeletal actin isoform genes within each species had 92.0 and 91.8% homology. From the cDNA sequences of the four α-skeletal actin isoforms in the two species, amino acid sequences of 377 amino acid residues were deduced. It was predicted that the two N-terminal amino acid residues of each protein are processed after translation. The amino acid sequences of α-skeletal actin 1 in the two Coryphaenoides species were identical, as were the amino acid sequences of α-skeletal actin 2 in the two species. The amino acid sequences of the two α-actin isoforms, α-skeletal actin 1 and α-skeletal actin 2, differed by only a single amino acid, Ala/Ser at the 155th position. Northern blot analysis showed that a similar amount of each of the two α-actin isoform mRNA was expressed in the skeletal muscle of the two Coryphaenoides species.