日本鬼鲉毒腺cDNA表达文库的构建和初步分析

以海洋生物日本鬼鲉毒腺为材料,应用SMART^TM cDNA Library Construction技术,构建以真核表达载体pcDNA3.0为基础的日本鬼鲉毒腺cDNA表达文库.通过对文库克隆的序列测定和初步生物信息学分析,获得94个日本鬼鲉毒腺新表达序列标签（ESTs）,其中已确定全长cDNA的克隆有35个,包括溶细胞素基因、类短链神经毒素蛋白、C型凝集素、巨噬细胞迁移抑制因子、致病相关基因等,这些基因在日本鬼鲉中绝大多数为首次发现.日本鬼鲉毒腺cDNA表达文库的成功构建,为深入研究日本鬼鲉毒腺的组分及其分子作用机理奠定了基础.     

性逆转石斑鱼性腺差异表达基因的克隆和筛选

以 17α 甲基睾丸酮 (17α MT)饲喂 2～ 4龄赤点石斑鱼 (Epinephelusakaara) ,成功地促使其性转变为具有生殖功能的雄鱼 .应用抑制性差减杂交 (SSH)技术构建了石斑鱼性反转前后性腺组织的SMARTcDNA文库及其cDNA差减文库 ,从中随机挑取 12 0 0个克隆进行了PCR和斑点杂交筛选 ,得到 12 0个差异表达cDNA片段 .挑选 71个cDNA克隆测序 ,将所测序列经GenBank检索和生物信息学比较 ,发现有 5 1个cDNA片段序列无明显的同源性 ,2 0个片段与报道的基因有较高同源性 .在这 2 0个具同源性的片段中有 3个片段可能是与性别分化密切相关的重要功能基因 ,它们是钙调蛋白基因、活性蛋白激酶C受体基因和一氧化氮合酶蛋白抑制剂基因 .这 3个基因被分别命名为鱼钙调蛋白基因 (GenBankaccession :AY2 8136 3)、鱼活性蛋白激酶C受体基因 (GenBankaccession :AY2 8136 4)和鱼一氧化氮合酶蛋白抑制剂基因 (GenBankaccession :AY2 8136 5 ) .     

中国蛇岛蝮蛇毒腺cDNA文库ESTs序列测定及生物信息学分析

前期我们构建了中国蛇岛蝮蛇(Gloydius shedaoensis shedaoensis,GSS)毒腺(GSSG)的cDNA(GSSG-cD-NA)文库。本文从构建的GSSG-cDNA文库阳性重组子中随机挑选了216个单克隆进行5’端表达序列标签(EST)单向测序,获得了211条高质量的ESTs。生物信息学序列比对分析结果表明84个克隆为已知功能基因,29个克隆为未知功能基因,98个克隆为新基因,分别占总ESTs的39.8%、13.7%和46.5%。成功获得了GSSG的部分ESTs序列,为GSS蛋白活性组分基因的克隆、表达和功能研究奠定了一定基础。     

新疆荒漠昆虫光滑鳖甲cDNA文库的构建及功能基因筛选

以过冬后早春的新疆荒漠拟步甲属昆虫光滑鳖甲(Anatolica Polita borealis)成虫为研究材料,应用SMARTTM cDNA Library Construction技术,通过总RNA提取,反转录合成cDNA,定向构建至噬菌体载体λTripEx2,经体外包装构建了光滑鳖甲的cDNA表达文库。测试结果表明库容量为2.2×106,重组率为84.8%。通过对cDNA文库克隆的序列测定和初步生物信息学分析,获得28个光滑鳖甲表达序列标签(ESTs),包括17个EST(60.7%)与NCBI中已注册的已知功能基因相似性较高,另外的11个EST(39.3%)则没有发现与之相似的序列,推测可能是功能未知的新基因。同时,利用PCR扩增文库技术从cDNA文库中克隆获得了光滑鳖甲的抗冻蛋白基因,初步表明光滑鳖甲cDNA表达文库构建的成功,为深入研究新疆荒漠昆虫的抗冻机理及发现新的昆虫功能基因奠定了基础。     

中华蜜蜂蜂毒镇静肽基因的cDNA克隆和表达

从中华蜜蜂 (Apisceranacerana)工蜂毒腺中快速抽提总RNA ,用RT PCR扩增得到大小约为2 5 0bp的cDNA片段 ,测序得到的片段长度为 2 34bp ,为蜂毒前镇静肽原 (preprosecapin)基因编码区的cDNA .以 3′RACE方法 ,扩增和测定了 3′端非编码区 2 19bp序列 .中蜂前镇静肽原cDNA序列与已报道的欧洲意蜂该基因cDNA序列具有 92 %同源性 ,氨基酸序列具有 87%同源性 .代表成熟肽镇静肽的最后 2 5个氨基酸序列 ,中蜂与意蜂同源性为 88% .3′端非编码区cDNA序列与欧洲意蜂序列有 73 1%同源性 .将中华蜜蜂蜂毒镇静肽成熟肽编码区与 3′非编码区部分克隆 ,构建了镇静肽与谷胱甘肽转移酶融合表达的载体pGEX AcSecapin .将载体转化大肠杆菌BL2 1(DE3)进行融合表达 .表达产物与抗GST抗体在 2 9kD处有很强的交叉反应 .大肠杆菌超声破碎后的上清液用SDS PAGE检测到表达的蛋白多为可溶性融合蛋白 ,通过亲和层析柱纯化和凝血酶的切割得到了镇静肽蛋白     

小鼠脂联素受体2基因编码区的克隆及序列分析

构建小鼠脂联素受体2(mAdipoR2)基因cDNA克隆,并进行序列及基因结构分析,为进一步研究小鼠AdipoR2的表达和生物学活性奠定基础。用RT—PCR方法扩增mAdipoR2基因cDNA,获得的片段连接至pGEM-T载体,转化JM109大肠杆菌,经酶切鉴定后,进行序列测定。利用因特网生物信息学资源分析mAdipoR2基因序列。结果成功地构建了mAdipoR2基因cDNA克隆,其序列与GenBank登录序列一致。通过与小鼠基因组序列比对,分析了mAdipoR2基因结构,其编码序列由7个外显子组成,编码1个含7个跨膜区的膜蛋白,但该受体不属于G蛋白偶联受体家族(GPCRs)。mAdipoR2与人及大鼠蛋白的同源性分别为91％和95％。     

无乳链球菌诱导吉富罗非鱼分泌型免疫球蛋白M(sIgM)重链基因的克隆及原核表达

以灭活的无乳链球菌(Streptococcus agalactiae)诱导后的吉富罗非鱼(Oreochromis niloticus)为材料,构建其头肾SMART cDNA文库,应用同源性克隆和RACE-PCR技术克隆到吉富罗非鱼(O.niloticus)分泌型免疫球蛋白M(sIgM)重链基因的全长cDNA序列并对其进行生物信息学分析。sIgM基因cDNA全长为1 921 bp,开放阅读框(ORF)为1 740 bp,5'端非编码区(5'-UTR)41 bp,3'端非编码区(3'-UTR)140 bp,编码579个氨基酸,N端有信号肽结构。预测分子量(MW)为64.26 kD,理论等电点(pI)为5.36。系统进化树分析显示,吉富罗非鱼(O.niloticus)sIgM与牙鲆(Paralichthys olivaceus)和军曹鱼(Rachycentron canadum)的亲缘关系较近。sIgM基因有4个恒定区。将吉富罗非鱼(O.niloticus)sIgM基因恒定区序列克隆到原核表达载体pET-28a(+)中,构建原核表达质粒pET28-sIgM,诱导表达后确定最优条件为37℃条件下,IPTG浓度为0.05 mmol/L时诱导4 h蛋白表达量最大。纯化蛋白后经Western blot分析,sIgM融合蛋白与鼠抗His-Tag发生特异结合,表明目的蛋白成功表达。     

大黄鱼Flotillin-1基因分子特征分析

浮舰蛋白-1(Flotillin-1)是属于SPFH家族的蛋白,是重要的脂筏标志性蛋白.在构建大黄鱼(Larimichthys crocea)肌肉组织cDNA文库的基础上,克隆了Flotillin-1基因,并进一步扩增出内含子.克隆到的序列全长为2497 bp,其中编码区1194 bp,编码397个氨基酸.生物信息学分析大黄鱼Flotillin-1有5类20个功能位点,存在2次跨膜结构,N端和C端都位于细胞膜内.大黄鱼Flotillin-1氨基酸序列具有非常高的保守性,与大西洋鲑和斑马鱼的同源性都在80%以上.在组织中的表达也非常广泛,其中在脾中的表达最强.     

巨型艾美耳球虫孢子化卵囊cDNA文库的构建

为构建巨型艾美耳球虫(Eimeria maxima)孢子倾卵囊cDNA表灰文库,从E.maxima孢子化卵囊中提取总RNA,以总RNA为模板、λTriplex2TM为栽体,利用SMARTTM cDNA文库构建试剂盒构建全长cDNA表达文库.经测定构建的E.maxima cDNA表达文库的原始文库容量为1×106 pfu/ml,扩增后的文库滴度为5×1010pfu/ml,重组率为95%,插入片断主要集中在0.5～1kb之间.根据已知序列设计引物,能从文库中扩增出编码E.maxima免疫球蛋白重链结合蛋白的基因序列片段.结果 表明所构建的E.maxima cDNA表达文库质量良好,为克隆、筛选E.maxima的功能性基因奠定了基础.     

玫瑰红绿海葵触手cDNA表达文库的构建和初步分析

海葵共有 10 0 0多种 ,栖息于世界各地的海洋中 ,从极地到热带、从潮间带到深海都有分布 ,我国地处温带和亚热带 ,海葵种类较多[1 ] 。海葵的单体呈圆柱状 ,柱体开口端为口盘 ,口盘周围环生众多触手 ,触手上有被称为刺丝囊的毒腺结构 ,能分泌毒液 ;柱体下端为基盘 ,海葵以其基盘吸附于甲壳、海藻等物体上 ,较少运动。海葵毒液成分主要是蛋白质和多肽 ,它们对甲壳类动物有较大毒性 ,对人或其它高等动物的毒性则相对较小。现代研究表明 ,海葵多肽毒素有多种药理效果 ,这些毒素主要作用于神经及心血管系统 ,可引起强心、降低血压、麻痹肌肉等多…     

Molecular characterization of an anther-preferential gene from rice

Expression levels of anther-expressed genes in rice were estimated by plaque hybridization. A total of 33 cDNAs, isolated randomly from an anther-enriched cDNA library, were used as probes to hybridize both anther and leaf cDNAs. The expression level of individual cDNA clones was then estimated by counting the number of plaques hybridized to each probe. Based on abundance patterns that appeared in both anther and leaf cDNA libraries, the clones were classified into three groups. This classification showed that the majority of the clones (one group) exhibited expression in both cDNA libraries at almost equal frequency. The other two groups showed either low or no expression in the leaf cDNA library. Among the cDNA clones,RA1003 (detected only in the anther cDNA library) was selected and further characterized at the molecular level. Consistent with the results of the plaque hybridization experiment, northern blot analysis also revealed no gene expression in vegetative organs, leaves, or roots. However, expression was high in the flowers, especially in the anthers. Detailed molecular studies of the gene are also described and discussed here.     

A study of cotyledon senescence in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) based on expressed sequence tags and gene expression

Cucumber cotyledons provide an excellent experimental system in which to investigate developmental changes in gene expression, from the early phase of heterotrophism through phototrophic growth to senescence. A cDNA library was prepared from the final stage of senescing cucumber cotyledons (<95% yellow) for studying the genes responsible for lipid mobilization during germination and senescence. This library had produced numerous senescence-associated clones in a previous study. Here, a total of 365 cDNA clones and their expression levels were examined via semi-quantitative RT-PCR. Up-regulation of expression was detected for several known and unknown genes. These results were used to investigate the possible functions for senescence-related genes during cotyledon development     

疣粒野生稻应答黄单胞杆菌水稻致病变种（Xoo）的基因芯片

探讨疣粒野生稻应答黄单胞杆菌水稻致病变种（Xoo）的基因芯片制作,通过芯片杂交筛选抗病相关基因。芯片含有2436个片段,来自于应答撖）O的疣粒野生稻差减文库和cDNA文库,通过芯片杂交及微阵列分析基因表达,选其中800个样品点测序比对。其中,35个无同源序列,大部分有同源序列的功能未知,已知功能的序列中明显上调表达的基因有：富含脯氨酸蛋白、泛素连接酶、伸展蛋白、谷胱甘肽S-转移酶II、脂类转移酶等,明显下调表达的基因有：细胞色素P450单加氧酶、醛缩酶、金属硫蛋白、硫氧还蛋白、热激蛋白等,表达无明显变化的基因有：抗坏血酸过氧化物酶、转铜伴侣、脂酶、花丝温敏H2A蛋白等。高通量基因芯片的利用及微阵列分析是筛选抗病相关基因、获取大量抗病相关信息的有效手段。     

Systematic isolation of genes expressed at low levels in inflorescence apices of Arabidopsis thaliana.

We constructed an equalized cDNA library from Arabidopsis inflorescence shoot apices including inflorescence meristem, floral meristem and flower tissue collected before stage 5 of flower development. The cDNA clones were arrayed on membranes and were differentially screened using cDNA pools from vegetative and inflorescence tissues as probes. Each clone was classified by expression specificity and expression level. By removing the clones that displayed hybridization signals, 384 out of 3264 clones in this library remained as candidates for inflorescence-specific mRNAs expressed at low levels. Sequence analysis of all selected clones indicated that 53 were identical and 120 were homologous to genes in public protein databases. The remaining 211 selected clones had no significant amino acid sequence similarities with those deduced from any reported genes, though 62 of them appeared in Arabidopsis expressed sequenced tags (ESTs). About 40% of the selected clones were novel, validating the present approach for gene discovery. Northern blot analysis of 22 randomly selected clones confirmed that most were expressed preferentially in inflorescence tissues. In addition, many clones were transcribed at relatively low levels. We demonstrate that the screening method of the present study is useful for systematic classification of cDNA species based on expression specificity.     

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.