光棘球海胆seali基因克隆及表达特性分析

seali基因隶属于PIWI超家族, 其编码的RNA结合蛋白在生殖细胞发育过程中发挥重要作用。研究经同源比对从光棘球海胆(Mesocentrotus nudus)性腺转录组数据库中筛选得到seali基因片段, 随后通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends, RACE), 获得其全长cDNA序列。光棘球海胆seali基因cDNA全长3462 bp, 其中3′UTR长度为416 bp, 5′UTR长度为180 bp, 其中3′UTR的加尾信号并非经典的AAUAAA或AUUAAA, 而是较少见的AAUACA。开放阅读框(Open Reading Frame, ORF)2862 bp, 编码954个氨基酸, 具有保守的PIWI和PAZ结构域, 多重序列比对和系统进化分析结果表明其属于Argonaute家族的PIWI亚家族成员。荧光定量PCR技术检测结果表明, Mnseali基因在光棘球海胆性腺、肠、管足和体腔细胞中均有表达, 在性腺中表达量最高。此外, Mnseali基因为母源因子, 在整个胚胎发育时期均有表达。在卵巢中, 随着卵母细胞的成熟, Mnseali的表达量逐渐升高, 而仅在成熟期的精巢中表达量显著上调。RNA原位杂交结果表明, Mnseali在光棘球海胆性腺的生殖细胞中特异表达, 是光棘球海胆生殖细胞标记基因。该研究为海胆生殖细胞发育相关的研究提供了支撑。     

人工繁殖虾夷马粪海胆生长模型

以虾夷马粪海胆(Strongylocentrotus intermedius)家系为实验材料,对比分析了每个母系从孵化后到17月龄(性成熟)的体重、壳径、壳高的生长情况,并进行了生长性状间的相关分析及预测模型的构建。结果表明:5~15月龄,各家系虾夷马粪海胆在壳径、壳高与体重方面的差异均不显著(P0.05),17月龄时,各家系的生长指标出现差异(P0.05);虾夷马粪海胆壳径、壳高生长呈线性模型,而体重生长呈指数模型;利用虾夷马粪海胆各月龄生长性状数据,进行了虾夷马粪海胆壳径、壳高与体重相关关系分析,R2值为0.68~0.86,而虾夷马粪海胆父母本的生长性状与子代的生长性状相关性较低(0~0.25)。虾夷马粪海胆体重、壳径和壳高的生长预测模型表明,通过11月龄体重预测17月龄体重的准确率可达73.6%;本研究有助于深入了解虾夷马粪海胆的生长发育特征,并为其健康养殖模式构建提供理论依据。     

南方鲇Vasa基因两种亚型cDNA的克隆及其表达

采用RT-PCR和RACE相结合的方法,从南方鲇分离到Vasa基因的两个亚型scVasa和scVaga-s。它们是同一基因在5′端经选择性剪接的产物,其cDNA全长分别为2525bp和2438bp,编码662和641个氨基酸。两者均具有DEAD-box家族成员特有的8个保守基序和Vasa的典型特征。南方鲇Vasa与银鲫相似性最高（73．3％）。两个亚型均特异地表达于雌雄性腺中。原位杂交结果表明：scVasa主要在卵巢Ⅰ、Ⅱ时相的卵母细胞和精巢的精原细胞和初级精母细胞中表达。半定量PCR结果显示,在生殖周期中,两种亚型在以Ⅱ时相卵母细胞为主体的卵巢恢复期表达均高于以Ⅲ-Ⅳ时相卵母细胞为主体的卵黄生成期[动物学报54（6）：1051—1060,2008]。     

光棘球海胆(Mesocentrotus nudus)TGF-β基因克隆及其对海水酸化的响应

旨在明确光棘球海胆(Mesocentrotus nudus)转化生长因子-β(transforming growth factorβ,TGF-β)基因的序列及结构信息,探明该基因在海水酸化胁迫下的表达模式。利用同源克隆和cDNA末端快速扩增(RACE)技术首次获得光棘球海胆TGF-β基因(命名为MnTGF-β)的全长cDNA序列。结果显示:(1)光棘球海胆TGF-β基因的cDNA全长为2 299 bp,其中5'非编码区长度为745 bp,3'非编码区长度为261 bp;开放阅读框(ORF)长度为1 290 bp,编码430个氨基酸,相对分子量为48.31 kD,理论等电点为5.34。(2)同源性及系统进化分析显示,光棘球海胆MnTGF-β蛋白序列与紫球海胆(Strongylocentrotus purpuratus)SpTGF-β2蛋白序列高度保守(同源性97.69%)。(3)实时荧光定量PCR(qRT-PCR)检测发现,MnTGF-β基因在光棘球海胆性腺、体腔液、肠、围口膜、管足5种组织中均有表达,其相对表达量从高到低依次为:管足围口膜肠体腔液性腺。(4)与自然海水组(pH 8.06±0.01)相比,当海水△pH为-0.3时,随酸化时间延长,MnTGF-β基因在光棘球海胆性腺中的相对表达量呈先升高而后极显著降低(P0.01)再极显著升高(P0.01)趋势,在管足中的相对表达量呈现极显著上调趋势(P0.01),而在肠组织中的相对表达量则先极显著降低(P0.01)而后显著升高(P0.05);当海水△pH为-0.4和-0.5时,随酸化时间的延长,MnTGF-β基因在光棘球海胆性腺和管足中的相对表达量呈现先降低后升高的趋势,而在肠组织中的相对表达量则呈现极显著降低趋势(P0.01)。     

虾夷扇贝脂多糖诱导的肿瘤坏死因子LITAF基因的克隆及表达分析

脂多糖诱导的肿瘤坏死因子(Lipopolysaccharide-induced TNF-alpha factor,LITAF)是一类重要的炎症细胞因子,在先天性免疫系统中发挥重要的介质作用。文章根据虾夷扇贝LITAF基因EST序列,应用RACE技术克隆了虾夷扇贝LITAF全长cDNA,对序列及编码的氨基酸进行生物信息学分析。结果显示,该基因cDNA全长1 551 bp,其5′非编码区包含76 bp,3′非编码区包含1 001 bp;开放阅读框(ORF)为474 bp,编码157个氨基酸,氨基酸序列中存在一个保守的LITAF结构域;理论分子量16.99 kDa,等电点为6.24。LITAF基因序列为3 698 bp,由3个外显子和两个内含子组成。利用实时荧光定量PCR技术分析LITAF在虾夷扇贝不同组织、不同胚胎发育阶段以及鳗弧菌(Vibrio anguillarum)刺激后各时间段的表达情况。结果表明:LITAF基因在所检测的6个成体组织中均有表达,其中肾脏的表达量最高;胚胎发育的7个时期中,担轮幼体时期表达量最高;菌刺激36 h实验组与对照组的表达量差异大。LITAF基因是LITAF家族的一员,推测LITAF基因参与虾夷扇贝的先天性免疫反应。     

虾夷扇贝β-actin基因的克隆和序列分析

为进一步研究虾夷扇贝功能基因的表达调控。利用SMART cDNA文库构建试剂盒成功构建了健康虾夷扇贝外套膜和肾脏两种组织的cDNA文库。对随机选取的4009个克隆进行5′端测序,比对,筛选出1条β肌动蛋白同源序列,对此EST序列两端进行扩增、测序,得到肌动蛋白基因cDNA全长序列。肌动蛋白基因cDNA全长1536bp(不包括polyA),5′端非编码区84bp,3′端非翻译区321bp,阅读框1131bp,编码377个氨基酸。在基因组DNA中,该基因被一个内含子分为两段,内含子位于第41和第42个氨基酸之间,长度为1498bp。系统发育分析显示该肌动蛋白属于β类型。本研究得到的虾夷扇贝β-肌动蛋白基因可以被用于作为定量某种虾夷扇贝mRNA的标准,这为继续研究虾夷扇贝其它功能基因,及其分子生物的进一步研究、促进其他相关分子发育和系统进化研究奠定了基础。     

MYP基因在中间球海胆及杂交海胆生殖腺不同发育时期的转录表达差异

摘要: 应用实时荧光定量PCR技术对主要卵黄蛋白(Major yolk protein, MYP)基因在中间球海胆和杂交海胆(中间球海胆♀×光棘球海胆♂)的生殖腺不同发育时期的转录表达差异进行了分析。结果表明, MYP基因在海胆雌雄个体生殖腺中转录水平上的表达差异不明显; MYP基因在中间球海胆生殖腺发育的4个时期的表达呈快速下降的趋势, 而在杂交海胆呈缓慢下降的趋势。在分析的生殖腺4个发育期中, 中间球海胆雌雄个体 MYP 基因的表达量(以18S rRNA为参照)分别从44.55%和41.17%下降到9.59%和1.83%; 杂交海胆的雌雄个体分别从37.66%和36.66%下降到19.22%和12.55%。MYP基因的转录表达量差异与杂交后生殖腺产生的变异密切相关。     

中华鳖vasa基因克隆及在卵母细胞中的表达分析

研究利用中华鳖为研究模型进行爬行类生殖细胞发育分化成熟等生物学研究,克隆了中华鳖vasa基因的cDNA序列,全长3865 bp,包括5'端非编码区90 bp,3'端非编码区1699 bp,开放阅读框长2076 bp,共编码691个氨基酸。中华鳖Vasa氨基酸序列包含DEAD-box家族蛋白8个保守保守功能域,在N末端有4个RGG重复序列和2个GG富集区,与小鼠Vasa蛋白的同源性较高（72%）。荧光定量PCR的结果表明,中华鳖vasa mRNA主要精巢和卵巢中表达,其他体组织中均难检测到表达。卵巢冰冻切片原位杂交结果显示:中华鳖vasa mRNA在生殖细胞中特异表达;在卵子发生过程中的不同发育期卵母细胞中呈现动态的变化。即vasa mRNA在初级卵母细胞及生长期卵母细胞中表达最强,且均匀分布在细胞质中,随着卵母细胞的逐渐增大,信号逐渐减弱,直至在成熟的卵母细胞中几乎检测不到表达信号,说明vasa可能在中华鳖早期卵母细胞发育中起重要作用。同时,vasa基因可作为中华鳖生殖细胞分子标记物,根据其mRNA的表达水平来鉴别不同发育时期的卵母细胞。研究结果为进一步开展中华鳖胚胎生殖细胞发育及配子生成,特别是研究中华鳖,乃至爬行类原始生殖细胞（Primordial Germ Cells,PGCs）的起源、迁移、分化等研究奠定了基础。     

金鱼PP2A-B′家族δ基因cDNA的克隆及表达分析

通过3′-RACE及5′-RACE技术克隆得到了金鱼蛋白磷酸酶2A(protein phosphatase2A,PP2A)调节亚基B′家族δ(Delta)基因的cDNA全序列.结果显示,金鱼δ基因cDNA全长2415bp,编码一个含555个氨基酸的蛋白.序列分析表明,该基因编码的蛋白与已知其他物种对应的B′家族蛋白质均有着很高的同源性.RT-PCR分析证明,该基因mRNA表达水平在大脑中为最高,肝脏、精巢、卵巢、肾脏和鳃中次之,鳍中最少.在不同胚胎时期中,两细胞期、多细胞期、囊胚期和原肠胚期表达最高,其他时期相对较低.在蛋白水平上,精巢、卵巢、大脑和心脏中最高,肝脏中次之,肾脏、鳃和鳍中最少.在胚胎中,两细胞期、多细胞期、囊胚期、原肠胚期和神经胚期及视原基期表达最高,脑泡分化和眼色素期表达量最少.由此可以推测PP2AB′-δ基因在金鱼不同组织和胚胎发育的不同时期中可能起着多种重要作用.     

金鱼PP2A-B′家族δ基因cDNA的克隆及表达分析

通过3′-RACE及5′-RACE技术克隆得到了金鱼蛋白磷酸酶2A（protein phosphatase2A,PP2A）调节亚基B′家族δ（Delta）基因的cDNA全序列.结果显示,金鱼δ基因cDNA全长2415bp,编码一个含555个氨基酸的蛋白.序列分析表明,该基因编码的蛋白与已知其他物种对应的B′家族蛋白质均有着很高的同源性.RT-PCR分析证明,该基因mRNA表达水平在大脑中为最高,肝脏、精巢、卵巢、肾脏和鳃中次之,鳍中最少.在不同胚胎时期中,两细胞期、多细胞期、囊胚期和原肠胚期表达最高,其他时期相对较低.在蛋白水平上,精巢、卵巢、大脑和心脏中最高,肝脏中次之,肾脏、鳃和鳍中最少.在胚胎中,两细胞期、多细胞期、囊胚期、原肠胚期和神经胚期及视原基期表达最高,脑泡分化和眼色素期表达量最少.由此可以推测PP2AB′-δ基因在金鱼不同组织和胚胎发育的不同时期中可能起着多种重要作用.     

Sea urchin actin gene subtypes. Gene number, linkage and evolution

The actin gene family of the sea urchin Strongylocentrotus purpuratus was analyzed by the genome blot method, using subcloned probes specific to the 3' terminal non-translated actin gene sequence, intervening sequence and coding region probes. We define an actin gene subtype as that gene or set of genes displaying homology with a given 3' terminal sequence probe, when hybridized at 55 degrees C, 0.75 M-Na+. By determining the often polymorphic restriction fragment band pattern displayed in genome blots by each probe, all, or almost all of the actin genes in this species could be classified. Our evidence shows that the S. purpuratus genome probably contains seven to eight actin genes, and these can be assigned to four subtypes. Studies of the expression of the genes (Shott et al., 1983) show that the actin genes of three of these subtypes code for cytoskeletal actins (Cy), while the fourth gives rise to a muscle-specific actin (M). We denote the array of S. purpuratus actin genes indicated by our data as follows. There is a single CyI actin gene, two or possibly three CyII genes (CyIIa, CyIIb, and possibly CyIIc), three CyIII actin genes (CyIIIa, CyIIIb, CyIIIc), and a single M actin gene. Comparative studies were carried out on the actin gene families of five other sea urchin species. At least the CyIIa and CyIIb genes are also linked in the Strongylocentrotus franciscanus genome, and this species also has a CyI gene, an M actin gene and at least two CyIII actin genes. It is not clear whether it also possesses a CyIIc actin gene, or a CyIIIc actin gene. The genome of a more closely related congener, Strongylocentrotus dr?bachiensis, includes 3' terminal sequences suggesting the presence of a CyIIc gene. In S. franciscanus and S. dr?bachiensis the first intron of the CyI gene has remained homologous with intron sequences of both the CyIIa and CyIIb genes, indicating a common origin of these three linked cytoskeletal actin genes. Of the four S. purpuratus 3' terminal subtype probe sequences only the CyI 3' terminal sequence has been conserved sufficiently during evolution to permit detection outside of the genus Strongylocentrotus. An unexpected observation was that a sequence found only in the 3' untranslated region of the CyII actin gene in the DNA of S. dr?bachiensis and S. purpuratus is represented as a large family of interspersed repeat sequences in the genome of S. franciscanus.     

Novel proteins belonging to the troponin C superfamily are encoded by a set of mRNAs in sea urchin embryos

The properties of several cDNA clones representing a family of mRNAs found in the embryonic ectoderm of Strongylocentrotus purpuratus are described. We have previously shown that these mRNAs (termed Spec for Strongylocentrotus purpuratus ectoderm) accumulate in the presumptive dorsal ectoderm of post-cleavage stage embryos and code for a group of 10 to 12 low molecular weight acidic proteins. We demonstrate here, using antibodies raised against the major Spec proteins, that the proteins are localized in the cytoplasm of dorsal ectoderm cells. Hybridization analysis and DNA sequencing show that the mRNAs coding for these proteins, although all related, can be divided into two subfamilies. Comparison of the translational reading frames of the Spec mRNAs with known protein sequences shows a significant homology with troponin C-related proteins, especially in the calcium-binding domains. We suggest that the Spec proteins are previously uncharacterized members of the troponin C superfamily.     

Complete Sequence of SpHox8 and Its Linkage in the Single Hox Gene Cluster of Strongylocentrotus purpuratus

SpHox8 is the paralog group 8 Hox gene of Strongylocentrotus purpuratus. This identification follows from an analysis of the sequence of the complete open reading frame of a late-gastrula-stage cDNA clone; from direct linkage to adjacent Hox genes in a fosmid contig; and from blot hybridizations carried out on pulse field gel electrophoretic separations. SpHox8 is a single-copy gene, and there is only one Hox gene cluster per genome in S. purpuratus. Received: 7 November 1996 / Accepted: 4 December 1996     

Characterization of five members of the actin gene family in the sea urchin

Hybridization of an actin cDNA clone (pSA38) to restriction enzyme digests of Strongylocentrotus purpuratus DNA indicates that the sea urchin genome contains at least five different actin genes. A sea urchin genomic clone library was screened for recombinants which hydridize to pSA38 and four genomic clones were isolated. Restriction maps were generated which indicate that three of these recombinants contain different actin genes, and that the fourth may be an allele to one of these. The restriction maps suggest that one clone contains two linked actin genes. This fact, which was confirmed by heteroduplex analysis, indicates that the actin gene family may be clustered. The linked genes are oriented in the same direction and spaced about 8.0 kilobases apart. In heteroduplexes between genomic clones two intervening sequences were seen. Significant homology is confined to the actin coding region and does not include any flanking sequence. Southern blot analysis reveals that repetitive DNA sequences are found in the region of the actin genes.     

Comparison of the bindin proteins of Strongylocentrotus franciscanus, S. purpuratus, and Lytechinus variegatus: sequences involved in the species specificity of fertilization.

Bindin is the sea urchin sperm acrosomal protein that is responsible for the species-specific adhesion of the sperm to the egg. Two new bindin cDNA sequences that contain the entire open reading frame for the binding precursor are reported: one for Strongylocentrotus franciscanus and one for Lytechinus variegatus. Both contain inverted repetitive sequences in their 3' untranslated regions, and the S. franciscanus cDNA contains an inverted repetitive sequence match between the 5' untranslated region and the coding region. The middle third of the mature bindin sequence is highly conserved in all three species, and the flanking sequences share short repeated sequences that vary in number between the species. Cross-fertilization data are reported for the species S. purpuratus, S. franciscanus, L. variegatus, and L. pictus. A barrier to cross-fertilization exists between the sympatric Strongylocentrotus species, but there is no barrier between the allopatric Lytechinus species.