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斑马鱼Hoxa-11a基因克隆及序列分析的研究浙江大学生物技术系薛良义和林凯先对斑马鱼基因组DNA进行研究。他们用PCR法从斑马鱼基因组DNA中扩增了HoXa-11a基因的完整编码序列,并将其克隆到PCR-ToPo载体,该基因包括两个外显子,其长度分别为622bp和233bp,其间的内含子为726bp,可共编码为284个氨基酸,它与人、鼠、鸡、爪蟾和矛尾鱼HoXa-11基因氨基酸序列的同源性分别为50.0%、51.3%、53.3%、56.7%和59.7%除同源异型盒外,外显子1区和内含子中也存在保守序列。外显子1中丙氨酸同类物与其两侧富含甘氨和丝氨酸亚区的积累是不同动物Hoxa-11…     

热带爪蟾Hoxa11基因克隆及序列的进化分析

 H oxa1 1基因是控制脊椎动物肢发育的重要基因 .根据人和鼠 H oxa1 1基因外显子 区和 区的保守序列设计了简并引物 ,采用 PCR法从热带爪蟾基因组 DNA中扩增和克隆到了H oxa1 1基因 ,并测定了核苷酸序列 .克隆的热带爪蟾 H oxa1 1基因片段长 1 598bp,由外显子 、内含子和外显子 三部分组成 ,其中外显子 60 4 bp,外显子 49bp.将该片段的核苷酸序列与人、鼠、斑马鱼 H oxa1 1基因的相应区域进行比较 ,发现该基因的内含子长度存在明显差异 .斑马鱼、热带爪蟾、鼠和人的内含子长度分别为 632 bp,945bp,1 42 1 bp和 1 41 2 bp,随动物进化阶梯的提高而变长 .外显子 区则高度保守 ,都是 49bp,外显子 区在长度上呈现约 1 0 %的变异 .将热带爪蟾 H oxa1 1基因编码的氨基酸序列与人、鼠、斑马鱼进行比较 ,它们之间分别有 67.0 %、66.5%和 46.0 %的同源性 .热带爪蟾与哺乳动物的同源性高于鱼类 ,可能反映了脊椎动物从鳍到肢的进化过程中 ,H oxa1 1基因经历了较多的变异 .     

大黄鱼肝表达抗菌肽2基因的克隆和原核表达

抗菌肽是在多种细胞中表达具有抗菌活性的肽类物质的总称,在免疫反应中发挥着非常重要的作用.通过同源克隆法克隆到大黄鱼肝脏表达的抗菌肽2(liver-expressed antimicrobial peptide-2,LEAP-2)基因的完整开放阅读框(Opening Reading Frame,ORF).克隆到的大黄鱼LEAP-2全长2236 bp,包含外显子Ⅰ78 bp,内含子Ⅰ880 bp,外显子Ⅱ179 bp,内含子Ⅱ1044 bp,外显子Ⅲ55 bp,编码序列312 bp,编码103个氨基酸.推断的氨基酸序列羧基端区域存在高度保守的4 个半胱氨酸残基,符合LEAP-2超家族的结构特征.同源性对比后显示LEAP-2基因在进化上高度保守,大黄鱼LEAP-2推断的氨基酸序列与牙鲆、黄颡鱼、蓝色鲶鱼和斑点叉尾鮰等鱼类之间的同源性均在95%以上.将大黄鱼LEAP-2 cDNA连接到pET-32a(+),构建了重组表达质粒pET-32a-LEAP-2,将其转化到大肠杆菌BL21上并用1.0 mmol/L IPTG诱导表达,获得了大小约为27 kDa的重组蛋白,与预期的一致.     

大黄鱼白细胞介素8基因克隆及特征分析

白细胞介素8是一种CXC型趋化性细胞因子,在免疫反应中起着非常重要的作用。本文在构建大黄鱼肌肉组织cD-NA文库的基础上,克隆了白细胞介素8基因。克隆到的白细胞介素8全长为2582bp,基因组包含106bp的5’端非编码区,52bp的外显子Ⅰ,168bp的内含子Ⅰ,133bp的外显子Ⅱ,149bp的内含子Ⅱ,87bp的外显子Ⅲ,682bp的内含子Ⅲ,13bp的外显子Ⅳ和1192bp的3’端非编码区,编码序列285bp,编码94个氨基酸。氨基酸序列具有趋化性因子CXC家族的结构特征,在进化上高度保守,与鲈鱼的同源性在90％以上。在检测的大黄鱼的10种组织中,表达量较高的为肾、肝、肠和脾,脑、心和肌肉中表达量较低。     

鹅掌楸LcUGE基因的克隆和组织表达分析

为了解鹅掌楸(Liriodendron chinense)的UGE基因功能,采用RACE和EPIC-PCR技术克隆到2个UGE基因,命名为LcUGE1和LcUGE2。结果表明,LcUGE1基因的c DNA全长为1 531 bp,包含1 050 bp的开放阅读框,编码349个氨基酸, gDNA长度为11 920 bp;LcUGE2基因的c DNA长度为1 378 bp,包含1 056 bp的开放阅读框,编码351个氨基酸,g DNA长度为6544 bp。LcUGE1和LcUGE2基因均含有9个外显子和8个内含子,且外显子长度和内含子剪切位点序列几乎一致,但内含子片段长度存在显著差异。编码的LcUGE1和LcUGE2蛋白高度保守,保守性达到82%。LcUGE1基因在雄蕊中表达量最高,而LcUGE2基因则在花萼中表达量最高。这表明LcUGEs基因可能参与鹅掌楸的生殖发育过程。     

翘嘴鲌胰岛素样生长因子-Ⅰ的克隆及序列分析

为探讨胰岛素样生长因子-Ⅰ(Insulin like growth factor-Ⅰ, IGF-Ⅰ)对于翘嘴鲌(Culter alburnus)生长性状的影响, 对其DNA序列进行了克隆。翘嘴鲌IGF-Ⅰ全长14567 bp, 由5个外显子和4个内含子组成。其5个外显子长度分别为298、160、182、36 和1360 bp。推测的阅读框为486 bp, 编码由161个氨基酸组成的IGF-Ⅰ前体蛋白。前体肽由信号肽、成熟肽、E肽三部分组成, 其中信号肽44个氨基酸, 成熟肽70个氨基酸, E肽47个氨基酸。成熟肽由B、C、A、D四个区域组成, 其中B结构域和A结构域的保守性最高, 在这2个区域包含由6个半胱氨酸残基形成的3个二硫键。翘嘴鲌B区域还包含保守的IGF-Ⅰ受体识别序列(PheB23-TyrB24-PheB25)。E肽的长度表明翘嘴鲌IGF-Ⅰ属Ea-2型。同源性分析表明翘嘴鲌与鲤科鱼类的IGF-Ⅰ编码氨基酸同源性较高, 为94%—100%, 但在聚类分析中翘嘴鲌并不是首先和鲌亚科的鱼类聚集在一起。Real-time qPCR组织特异性表达结果显示IGF-Ⅰ mRNA在肝脏组织中的表达量最高, 脾、心脏、精巢、脑次之, 肾、鳃、胃和卵巢中表达量较低。翘嘴鲌IGF-Ⅰ基因4个内含子长度分别为1170、9364、251 和1746 bp。相对外显子来说, 种间内含子变异较大, 其中第三内含子变异最大。翘嘴鲌IGF-Ⅰ基因中包含6个微卫星, (GATG)₅AATAT (ATAG)₁₁位于第一内含子中, (CT)₈、(TTA)₅、(AC)₁₃、(TG)₁₂和(ATT)₅位于第二内含子中。其中4个微卫星位点具有多态性, 将它们在120尾同塘养殖的翘嘴鲌中进行基因型与生长性状的关联性分析, 均未达到显著水平(P>0.05)。结果为进一步研究该基因的表达、功能及其转录调控特征奠定了分子基础。     

宽鳍鱲胰岛素样生长因子-Ⅰ的克隆及繁殖期前后表达分析

为探讨胰岛素样生长因子-Ⅰ(Insulin like growth factor-Ⅰ, IGF-Ⅰ)对宽鳍鱲(Zacco platypus)繁殖期前后生长性状的影响, 开展了宽鳍鱲IGF-Ⅰ基因序列的克隆及表达定位分析。宽鳍鱲IGF-Ⅰ基因全长13707 bp, 包含5个外显子、4个内含子, 其中5个外显子长度分别为222、160、182、36和829 bp; 内含子长度分别为1194、7771、254和1879 bp, 推测开放阅读框为486 bp, 编码161个氨基酸。实时荧光定量PCR(Real-time qPCR, RT-qPCR)结果显示, 宽鳍鱲IGF-Ⅰ mRNA在肝脏组织中的表达水平最高, 其次是性腺、脾脏、心脏和脑, 在肾脏中表达水平最低。在7—8月, 处于繁殖期的性成熟宽鳍鱲性腺中IGF-Ⅰ表达水平显著上升, 繁殖期过后回落至最低值。在其他组织中IGF-Ⅰ表达水平在生长发育过程和繁殖期前后波动不大, 且雄鱼大多数组织中IGF-Ⅰ基因平均表达水平高于雌鱼。荧光原位杂交技术(Fluorescence in situ hybridization, FISH)定位显示, 宽鳍鱲IGF-Ⅰ基因基本为胞浆阳性, 少数为核阳性, 在肝组织中呈全胞质性分布, 在性腺组织的精母细胞、卵泡膜及卵泡液中阳性表达。上述结果表明宽鳍鱲IGF-Ⅰ基因表达模式具有性别差异性, 推测精巢中IGF-Ⅰ在繁殖期的高表达是宽鳍鱲雄性成体大于雌性成体的原因之一。研究结果为宽鳍鱲的性二态和人工繁育的研究提供参考资料。     

鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法,克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明,鳜鱼Try基因cDNA全长为896 bp,其中开放阅读框 (open reading frame,ORF)为744 bp,编码247个氨基酸. 序列同源性分析发现,鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp,其中ORF为1 539 bp,编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明,鳜鱼Try基因由4个内含子和5个外显子组成,全长1 362 bp;鳜鱼Amy基因由8个内含子和9个外显子组成,全长4 267 bp;鳜鱼Pep基因由8个内含子和9个外显子组成,全长 4 032 bp,与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列,并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究,为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.     

草鱼与鲢鱼肥胖基因克隆与序列分析

肥胖基因产物leptin是调节哺乳动物摄食、能量代谢等生命活动的重要细胞因子。 应用RT-PCR和RACE法获得了草鱼(Ctenopharyngodon idellus)和鲢鱼(Hypophthalmichthys molitrix) leptin基因的全长cDNA序列分别为1 096 bp和1 176 bp,编码173和172个氨基酸。氨基酸序列同源性分析表明,草鱼和鲢鱼的leptin序列与其它鲤科鱼类leptin的同源性较高,而与其他鱼类的leptin同源性很低,但所有鱼类的leptin均含有用于形成二硫键的高度保守的半胱氨酸。系统进化树分析显示,草鱼和鲢鱼leptin与其他鱼类leptin聚于一进化分支。应用PCR和Genome Walker方法,进一步获得了草鱼和鲢鱼leptin基因的内含子和5′侧翼区序列。结果表明,获得的草鱼和鲢鱼leptin基因长度分别为2 129 bp和2 192 bp,含有与其他脊椎动物leptin相似的基因结构（含三个外显子和两个内含子）。本研究为深入研究鱼类肥胖基因结构功能关系与鱼类抗肥胖品系定向遗传选育奠定了良好的基础。     

马哈利樱桃PGIP cDNA克隆序列分析

以马哈利樱桃（Prunus mahaleb L.）为材料,通过RT－PCR获得了1045bp的目的段,经克隆测序,证实该片段包含1个完整的开放阅读框架,该阅读框架由990碱基组成,编码330个氨基酸。该序列与杏、梨、苹果的PGIP cDNA序列同源性分别达97.2%、83.4%和83.6%,可能编码的氨基酸与杏、梨、苹果的PGIP cDNA所编码的氨基酸的同源性分别达到96.7%、85.2%和85.2%。与已经克隆的PGIP DNA序列的对比分析表明,PGIP DNA序列中包含2个外显子和1个内含子,内含子全长147 ,符合TG－AG规律,2个外显子长度分别为581bp、464bp。     

Structure and expression of the mouse prealbumin gene

We cloned a genomic DNA fragment which covers the entire sequence of the mouse prealbumin gene and then studied the structure. The coding regions are separated into four exons by three introns, and these numbers, the sizes of the exons and the relative sites of the exon-intron junctions are all in complete agreement with those determined for the human gene. The sequences of four exons can be aligned perfectly with that of the previously determined mouse prealbumin cDNA. In addition to the exon regions, we found two highly conserved DNA regions between the mouse and human prealbumin genes, one in the 5'-flanking region of the gene and the other in the 3' end region of the first intron. These DNA regions contain several consensus glucocorticoid receptor-binding site sequences, and the latter also contains an enhancer sequence present in the immunoglobulin kappa-chain joining-constant kappa intron. RNA hybridizing to the mouse prealbumin cDNA was detected in the extracts from liver, brain, and kidney, but was not detected in testes, spleen, or heart. Little change was caused in the level of prealbumin mRNA in the liver by administration of dexamethasone to mice.     

Isolation, chromosomal localization, and nucleotide sequence of the human HOX 1.4 homeobox

We have isolated a 14-kb DNA sequence containing a single homeobox from a low-stringency screen of a human genomic phage library by using heterologous homeobox sequences as probes. Chromosomal mapping of this clone using in situ hybridization to metaphase chromosomes and a panel of mouse x human somatic cell hybrids localized it to human chromosome 7p13-p15 in the region of the HOX 1 locus. We have sequenced the homeobox and show it has 100% identity to the deduced amino acid sequence of the mouse Hox-1.4 homeobox. We detect no restriction fragment length polymorphisms with the 14-kb clone, which is devoid of any moderately repetitive DNA sequences. This implies an inability of this region to tolerate change in sequence, consistent with a function highly conserved throughout evolution. The regions in the human genome where homeobox-containing loci reside share patterns of organization and sequence and have other gene loci in common, implying evolutionary constraints over these regions and providing clues on how they may have evolved.     

Structures and chromosomal localizations of two human genes encoding synaptobrevins 1 and 2

Synaptobrevins 1 and 2 are small integral membrane proteins specific for synaptic vesicles in neurons. Two cosmid clones containing the human genes encoding synaptobrevins 1 and 2 (gene symbols SYB1 and SYB2, respectively) were isolated and characterized. The coding regions of the synaptobrevin genes are highly homologous to each other and are interrupted at identical positions by introns of different size and sequence. Each gene is organized into five exons whose boundaries correspond to those of the protein domains. Exon I contains part of the initiator methionine codon whereas exon II encodes the variable and immunogenic amino-terminal domain of the synaptobrevins. The third exon comprises the highly conserved central domain of the synaptobrevins, exon IV encodes most of the transmembrane region, and exon V contains the last residues of the transmembrane region and the small intravesicular carboxyl terminus. Comparisons of the synaptobrevin sequences in five species from Drosophila with man indicate a selective conservation of sequences adjacent to the synaptic vesicle surface, suggesting a function at the membrane-cystosol interface. The chromosomal localizations of the human and mouse SYB1 and SYB2 genes were determined using hybrid cell lines. SYB1 was localized to the short arm of human chromosome 12 and to mouse chromosome 6 whereas SYB2 was found on the distal portion of the short arm of human chromosome 17 and on mouse chromosome 11. A PstI restriction fragment length polymorphism was identified at the SYB2 locus.     

Genomic organization of the human CD5 gene

CD5 is a member of the family of receptors which contain extracellular domains homologous to the type I macrophage scavenger receptor cysteine-rich (SRCR) domain. Here, we compare the exon/intron organization of the human CD5 gene with its mouse homologue, as well as with the human CD6 gene, the closest related member of the SRCR superfamily. The human CD5 gene spans about 24.5 kb and consists of at least 11 exons. These exons are conserved in size, number, and structure in the mouse CD5 homologue. No evidence for the biallelic polymorphism reported in the mouse could be found among a population of 100 individuals of different ethnic origins. The human CD5 gene maps to the Chromosome (Chr) 11q12.2 region, 82 kb downstream from the human CD6 gene, in a head-to-tail orientation, a situation which recalls that reported at mouse Chr 19. The exon/intron organization of the human CD5 and CD6 genes was very similar, differing in the size of intron 1 and the number of exons coding for their cytoplasmic regions. While several isoforms, resulting from alternative splicing of the cytoplasmic exons, have been reported for CD6, we only found evidence of a cytoplasmic tailless CD5 isoform. The conserved structure of the CD5 and CD6 loci, both in mouse and human genomes, supports the notion that the two genes may have evolved from duplication of a primordial gene. The existence of a gene complex for the SRCR superfamily on human Chr 11q (and mouse Chr 19) still remains to be disclosed.