非洲爪蟾基因MGC64236的克隆及初步分析

MGC64236基因是本实验室用脐静脉内皮细胞免疫的兔血清筛选非洲爪蟾cDNA文库而鉴定的一个功能未知的基因.本研究提取非洲爪蟾受精卵总RNA通过RT-PCR得到基因MGC64236的开放读码框651 bp、编码202个氨基酸;运用生物信息学研究工具进行分析,发现该基因编码的蛋白有3个潜在的跨膜域,有一保守的结构域DUF1370, 可能通过其胞内部分的磷酸化机制在介导细胞内外的信号转导中发挥重要作用;在非洲爪蟾胚胎各个发育时期用RT-PCR检测该基因的表达情况,发现在非洲爪蟾胚胎发育的几个重要时期该基因都有高表达,而在成体则特异地表达于脑和眼等神经组织;构建绿色荧光融合蛋白真核表达载体并转染HEK293细胞, 对MGC64236蛋白的亚细胞定位,发现MGC64236蛋白比较特异地表达在细胞膜.     

非洲爪蟾中一种长型肽聚糖识别蛋白的克隆与表达分析(英文)

肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)是固有免疫系统中一类重要的模式识别受体。该文首次从两栖类模式生物-非洲爪蟾(Xenopus tropicalis)中克隆得到了一个长型PGRP(XtPGRP-L)基因。XtPGRP-L具有5个外显子和4个内含子的基因组结构,该结构在进化的过程中比较保守。序列比对与系统进化分析显示XtPGRP-L具有保守的酰胺酶活性位点。蛋白质建模显示XtPGRP-L拥有保守的3-D结构。实时定量PCR检测显示,XtPGRP-L在非洲爪蟾胚胎早期不表达,到72h蝌蚪期开始表达。在成体的肝脏、肺、肠和胃高表达。同时,在LPS刺激后,XtPGRP-L在肝脏、肠和胃中呈明显上调表达。结果表明,XtPGRP-L在非洲爪蟾固有免疫系统中可能具有重要的作用。     

非洲爪蟾BAFF及其信号通路相关基因的比较生物信息学分析

目的:对非洲爪蟾BAFF和BAFF信号通路相关基因进行了分析.方法:采用生物信息学方法对两栖类重要的模式生物-非洲爪蟾的基因组和EST数据库进行分析.结果:非洲爪蟾BAFF cDNA全长为557 bp,编码218个氨基酸.与人BAFF序列相似性为37.5％.该文一共得到了14个BAFF信号通路相关基因.通过与人BAFF信号通路进行比较,对非洲爪蟾这14个BAFF信号通路相关基因进行了分析.结论:BAFF和BAFF信号通路在进化过程中较为保守,这为进一步研究低等脊椎动物BAFF功能和信号通路具有重要的指导作用.     

非洲爪蟾在神经系统疾病研究中的应用

在生物医学研究领域,使用合适的动物模型研究大脑运行机制和疾病机理至关重要。作为经典的脊椎动物模型,非洲爪蟾在研究神经环路构建和功能以及神经疾病的分子机制中具有一定优势。因为非洲爪蟾的胚胎发育过程与人类器官形成过程相似,同时,神经系统疾病往往与神经系统功能异常和发育缺陷有关,所以一些人类疾病在基因和形态上的功能机制可以通过非洲爪蟾的胚胎发育模型进行在体研究,且神经前体细胞的增殖分化以及视觉刺激依赖的神经环路稳态和功能研究已经相对成熟。目前,该模型已经在癫痫和自闭症及表观遗传调控等相关疾病的研究上得到了应用。该文将分别介绍非洲爪蟾蝌蚪模型在环境毒物诱发疾病、神经发育障碍以及表观遗传疾病机制研究领域的最新进展。     

Sox1基因在爪蟾早期发育中的时空表达图式

克隆了非洲爪蟾的Sox1基因并研究了它在非洲爪蟾早期发育过程中的时空表达图式,比较了Sox1—3基因在发育的脑和眼中的表达图式。序列比对分析显示Sox1—3蛋白在其HMG框结构域具有高度的保守性。通过RT-PCR方法分析了Sox1基因在爪蟾早期不同发育时段的表达情况,结果显示Sox1基因从未受精卵到尾芽期均有表达,但表达强度有所差异。原位杂交结果显示,在早期卵裂阶段和囊胚期,Sox1基因主要在动物极表达;从神经板期开始,Sox1基因主要在中枢神经系统和眼原基中表达。在蝌蚪期,Sox1与Sox2、Sox3在脑部和眼睛的表达区域有所不同。对于爪蟾Sox1基因时空表达图式的研究将有助于阐明SoxB1基因家族在脊椎动物神经系统发生过程中的作用。     

非洲爪蟾早期胚胎发育中的Wnt信号

非洲爪蟾是脊椎动物胚胎发育研究中的几种重要模式生物之一,为揭示早期胚胎发育中的分子调控机制做出了显著的贡献.其中一个重要的发现就是细胞信号通路在胚胎发育中起到非常关键的调控作用.本文简单介绍Wnt信号在爪蟾早期胚胎发育不同时期的几种调控作用.     

日本蟾蜍聚集素cDNA的克隆与序列分析

为研究蟾酥、蟾衣、蟾皮中多肽类有效成分,通过菌落PCR对日本蟾蜍(Bufo japonicus formosus)皮肤cDNA质粒文库进行了筛选,获得了聚集素(clusterin,CLU)全长cDNA序列(GenBank登录号为JX035891)并对其进行了生物信息学分析。日本蟾蜍clucDNA全长为1 616 bp,包括1 332 bp的开放阅读框(open reading frame,ORF)、5’端30 bp及3’端254 bp的非翻译区(untranslated region,UTR)。根据cDNA序列推导的日本蟾蜍CLU前体蛋白由443个氨基酸残基组成,其中含20个氨基酸残基组成的信号肽,6个糖基化位点,10个可形成二硫键的半胱氨酸残基和2个卷曲螺旋区域。氨基酸序列同源性分析显示,日本蟾蜍CLU与非洲爪蟾(Xenopus laevis)的同源性为65%,与其他7种动物的同源性则介于41%-48%之间。     

免疫代价不是非洲爪蟾语音性信号真实度的维持机制

性选择理论认为,雄性性信号的真实度是雌性根据雄性表型质量进行配偶选择的前提。而维持这种真实度的机制普遍被认为是基于障碍原理,即性信号的表达必须付出与身体质量关联的存活代价,性信号与存活代价在个体内呈负的权衡关系,在个体间呈正相关关系。已有研究表明,免疫能力是表达性信号潜在的存活代价。但免疫代价在维持无尾两栖类语音性信号真实度中的作用尚不清楚。以非洲爪蟾为研究对象,在能量限制条件下采用灭活的大肠杆菌激活其免疫系统,测定鸣叫时长及相关繁殖行为表现,检验免疫能力与语音性信号表达间是否存在生理权衡。结果表明,免疫激活和能量限制对非洲爪蟾鸣叫时长和趋声行为都无影响;温度变化对鸣叫时长有显著作用。这些结果提示:非洲爪蟾语音性信号表达和免疫能力间不存在生理权衡,免疫代价不是非洲爪蟾语音性信号真实度的维持机制。     

乙酰胆碱受体α4β2亚型在非洲爪蟾卵母细胞中的表达

通过建立乙酰胆碱受体α4β2亚型(α4β2 nAChR)在非洲爪蟾卵母细胞中的表达模型,以便以α4β2 nAChR为作用靶点进行药物筛选。将乙酰胆碱受体亚基基因α4、β2体外转录获得的cRNA,通过显微注射的方法注入非洲爪蟾卵母细胞,并对该受体的表达情况进行检测。结果显示,乙酰胆碱受体α4β2亚型在蛙卵细胞中获得了有效表达,记录到典型的ACh配体门控的离子通道内向电流。     

移植非洲爪蟾胚胎细胞核引起花背蟾蜍成熟卵单性发育的研究

将非洲爪蟾胚胎细胞核移入花背蟾蜍成熟未受精卵后,得到了发育至各期的胚胎。对发育不同的时期的胚胎,进行了乳酸脱氢酶同工酶谱及染色体组型分析,结果一显示其均与受体一致。根据实验分析认为,非洲爪蟾的胚胎细胞进入花背蟾蜍成熟卵后,引起的是花背蟾蜍的单性发育。     

小菜蛾PGRP-S2基因的克隆表达及功能分析

肽聚糖识别蛋白(peptidoglycan recognition protein,PGRP)对于昆虫来说是一种高度保守的病原识别蛋白。为阐明PGRP-S2在小菜蛾Plutella xylostella抵抗病原微生物过程中的作用,本研究结合RT-PCR和RACE技术克隆得到小菜蛾PGRP-S2基因的cDNA全长序列,命名为PGRP-S2(GenBank登录号:MG570190)。生物信息学分析结果表明,PGRP-S2的开放阅读框为588 bp,编码195个氨基酸;蛋白质预测分子量为21.46 kDa,理论等电点为8.46;编码蛋白具有PGRP超家族保守结构域和酰胺酶结构域,是典型的肽聚糖识别蛋白,包含一条信号肽,不存在跨膜结构;同源序列比对和系统进化树分析表明PGRP-S2与家蚕Bombyx mori的BmPGRP-S 1进化距离最近。利用大肠杆菌Escherichia coli BL21(DE3)高效表达重组蛋白PxPGRP-S2,利用倒置显微镜及平板涂布观察重组蛋白对大肠杆菌E.coli和金黄色葡萄球菌Staphylococcus aureus的作用,结果表明PxPGRP-S2蛋白能够与两种细菌发生结合并凝集细菌,但不具备直接杀菌功能。本研究为进一步研究基于PGRP-S2介导的小菜蛾免疫防御反应提供基础。     

Molecular cloning, expression analysis of insulin-like growth factor I (IGF-I) gene and IGF-I serum concentration in female and male Tongue sole (Cynoglossus semilaevis)

Insulin-like growth factor I (IGF-I) is a polypeptide hormone that regulates growth during all stages of development in vertebrates. To examine the mechanisms of the sexual growth dimorphism in the Tongue sole (Cynoglossus semilaevis), molecular cloning, expression analysis of IGF-I gene and IGF-I serum concentration analysis were performed. As a result, the IGF-I cDNA sequence is 911 bp, which contains an open reading frame (ORF) of 564 bp encoding a protein of 187 amino acids. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. The IGF-I mRNA was predominantly expressed in liver, and the IGF-I expression levels in females and extra-large males were 1.9 and 10.2 times as much as those in normal males, respectively. Sex differences in IGF-I mRNA expressions at early life stages were also examined by using a full-sib family of C. semilaevis, and the IGF-I mRNA was detected at all of the 27 sampling points from 10 to 410 days old. An increase in IGF-I mRNA was detected after 190 day old fish. The significantly higher levels of IGF-I mRNA in females were observed after 190 days old in comparison with males (P < 0.01). The IGF-I concentrations in serum of mature individuals were detected by ELISA. The IGF-I level in the serum of females was approximately two times as much as that of males. Consequently, IGF-I may play an important role in the endocrine regulation of the sexually dimorphic growth of C. semilaevis.     

三角帆蚌热休克蛋白70基因克隆及其表达分析

对三角帆蚌HSP70基因序列进行全长克隆及其分子生物学分析,并检测其在不同水温刺激下鳃组织中的表达变化。通过高通量转录组测序获得三角帆蚌HSP70基因（HcHSP70）长片段,采用3'RACE对其进行了3'末端克隆,经拼接得到HcHSP70 cDNA全长序列。采用多种分子生物学软件对HcHSP70 cDNA全长序列进行了特征分析,采用实时荧光定量PCR技术检测了其组织分布,并结合Western-blot技术检测蚌鳃中该基因mRNA与蛋白经不同水温刺激后的表达变化。结果显示,HcHSP70 cDNA全长为2298 bp,其中开放阅读框为1974 bp,编码657个氨基酸。预测分子量大小为71.6 Ku,pH7.0时的理论等电点为5.61。氨基酸序列分析表明,HcHSP70氨基酸序列含HSP70家族的3个标签序列（I₉DLGTTYS₁₆、I₁₉₇FDLGGGTFDVSIL₂₁₀和I₃₃₆ VLVGGSTRIPKVQK₃₅₀）,与长牡蛎及泥蚶的HSP70同源性最高（91%）。实时荧光定量PCR检测结果显示,HcHSP70在鳃、性腺、肝胰腺、外套膜及肌肉等5种被检组织中均有表达,以肝胰腺中的表达水平最高。实时荧光定量PCR与Western-blot技术检测皆表明,蚌鳃组织中HcHSP70基因与蛋白的表达量在37℃时达到最高,而在40℃水温刺激下表达水平下调至正常值,表明其在适应高温刺激时发挥了重要作用。     

ANALYSIS OF THE Vitellogenin GENE OF RICE MOTH,Corcyra cephalonica STAINTON

Vitellogenin (Vg) is a precursor of the major yolk protein, an essential nutrient for the embryonic development of oviparous animals including insects. Here, the gene(CceVg [Corcyra cephalonica Vg] ) encoding the Vg (CceVg of moth, C. cephalonica, was cloned and sequenced. The gene sequence was 6,721‐bp long and contained 5five introns and six exons that together formed a 5,382‐bp open reading frame. The deduced protein (CceVg) consisted of 1,793 amino acid residues, including a 16‐amino‐acid signal peptide. The putative molecular weight of the primary Vg protein was 202.46 kDa. The CceVg contained all conserved domains and motifs that were commonly found in most insect Vgs except the presence of a polyserine tract at the C‐terminal region, which had not been reported in other lepidopteran Vgs. The expression pattern showed thatCceVg was first transcribed at a very low level in the early larval stage but disappeared in later stage larva. In female, theCceVg mRNA was detected in early pupal stage and throughout adult stage. Interestingly, theCceVg mRNA was detected only in mated males at low levels, not in the virgin ones. Injection ofCceVg double‐stranded RNA into early‐emergent females caused severely abnormal ovaries.     

Molecular cloning and the nucleotide sequence of cDNA to mRNA for non-neuronal enolase (alpha alpha enolase) of rat brain and liver.

The nucleotide sequence for alpha alpha enolase (non-neuronal enolase: NNE) of rat brain and liver was determined from recombinant cDNA clones. The sequence was composed of 1722 bp which included the 1299 bp of the complete coding region, the 108 bp of the 5'-noncoding region and the 312 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, the poly(A) tail was also found. A potential ribosome-binding site was located 30 nucleotides upstream to the initiation codon in the 5'-noncoding region. The amino acid sequence deduced from the nucleotide sequence was 433 amino acids in length and showed very high homology (82%) to the amino acid sequence of gamma gamma enolase (neuron-specific enolase: NSE), although the nucleotide sequence showed slightly lower homology (75%). The size of NNE mRNA was approximately 1800 bases by Northern transfer analysis and much shorter than that of NSE mRNA (2400 bases) indicating a short 3'-noncoding region. A dot-blot hybridization and Northern transfer analysis of cytoplasmic RNA from the developing rat brains using a labeled 3'-noncoding region of cDNA (no homology between NSE and NNE) showed a decrease of NNE mRNA at around 10 postnatal days and then a gradual increase to adult age without changes of mRNA size. Liver mRNA did not show any significant change during development.