LDL受体基因内含子15结构分析及其在家族性高胆固醇血症基因诊断中的应用

为了解人类ＬＤＬ受体基因内含子１５的遗传背景,利用长链ＰＣＲ和锚定ＰＣＲ分离了ＬＤＬ受体基因外显子１５－内含子１５－外显子１６和内含子１５的３‘末端片段。利用Ｄｙｎａｌｂｅａｄｓ固相单链分离ＰＣＲ产物直接测序法测定了内含子１５ ３’末端１２２２个碱基序列。序列显示：３‘末端含有由１６个碱基组成的典型３’末端剪接位点;３‘端上游第３１个碱基处含有经典分支位点,除了经典分支位点外,在３’末端上游第２０     

中华绒螯蟹蜕皮抑制激素1(Ers-MIH1)基因组DNA的分子克隆和序列分析

运用反向PCR (IPCR)技术首次克隆得到全长为 3 50 6bp的中华绒螯蟹 (Eriocheirjaponicasinensis)蜕皮抑制激素 1(MIH 1)基因组DNA序列 (GenBank检索号 :AY3 10 3 13 )。该序列包括 3个外显子、 2个内含子、 412bp的 5′端上游调控区和 917bp的 3′端UTR。编码区的第 1个内含子将信号肽分开 ,第 2个内含子将成熟肽分开。MIH 1基因的外显子和内含子接头区符合受体拼接点和供体拼接点的GT AG法则。MIH 1基因412bp的 5′端侧翼区含有和其它真核基因相似的启动子元件 ,即包括与其它节肢动物高度相似的起始子、TATA盒以及cAMP效应元件结合蛋白的结合位点序列。中华绒螯蟹MIH 1基因的组织方式与斑纹和食用黄道蟹的MIH基因相同。推导的多肽由 75个氨基酸的成熟肽和 3 5个氨基酸的信号肽组成 ,成熟肽的氨基酸序列和食用黄道蟹、三叶真蟹及美洲黄道蟹的一致性在 64% -65%之间     

牛催乳素基因组及其cDNA全长序列的分子克隆和分析

通过LongPCR等技术首次克隆得到全长9388bp的牛催乳素（bPRL)基因组序列（GenBank登录号AF426315）,其中包括bPRL基因全部5个外显子和4个内含子,5′端854bp的上游调控区以及3′端69bp的UTR,AF426315基因编码的蛋白质在GenBank中的序号为AAL28075,由229个氨基酸残基组成,1-30位氨基酸残基为信号肽序列,成熟的多肽含有199个氨基酸残基,将bPRL基因组DNA真核表达载体转染COS-7细胞后通过RT-PCR得到长度为804bp的bPRLcDNA序列,该序列涵盖了bPRL基因的全部ORF区,证明本研究所获得的bPRL基因组DNA具有转录的生物学功能,Blast搜索结果显示,GenBank数据库中收集有多条bPRL基因的mRNA和EST序列,各序列间存在多个SNP位点,主要分布于下游编码区和3′端的UTR,这些位点均未改变相应的氨基酸残基的性质,此外,5′端编码信号肽序列的区域呈现高度保守性。     

褐飞虱胰岛素酶基因的结构特点研究

胰岛素酶通过影响胰岛素的含量在稻飞虱翅型分化中起到重要的调控作用。本研究借助已获得的白背飞虱胰岛素酶的部分氨基酸序列,成功从褐飞虱转录组数据库中比对到带有褐飞虱胰岛素酶基因的c DNA序列,该序列含有一个带有起始密码子ATG和终止密码子TGA的开放阅读框,全长为1425 bp,编码的蛋白质具有胰岛素酶的典型结构:Peptidase_M16超家族和Peptidase_M16_C超家族。再将开放阅读框序列与褐飞虱基因组序列进行比对,得到胰岛素酶基因全长为1 674 bp,含有5个外显子和4个内含子。这是首次有关褐飞虱胰岛素酶基因结构的详细报道,为该基因的体外表达和功能研究打下了良好基础,将有利于探索该酶在稻飞虱翅型分化中的具体调控机制。同时本研究还发现胰岛素酶基因上游320 bp至858 bp处存有一个CpG岛区域,由于甲基化通常发生在基因上游的CpG岛区域,从而调控基因的表达,因此推测这个CpG岛区域可能存在甲基化现象,且在不同翅型的飞虱中甲基化水平存在差异,从而影响翅型分化。这一推测一旦获得实验验证,将有助于深入了解稻飞虱翅型分化的机制。     

水稻谷氨酰半胱氨酸合成酶基因的结构和表达分析

利用该实验室T-DNA标签的编号为L395的水稻突变体,克隆了一个编码水稻谷胱苷肽(GSH)合成途径中关键酶即谷氨酰半胱氨酸合成酶(GCs)的基因,将其命名为OsGCS(Genbank accession No.AJ508915).该基因位于水稻第五染色体上,OsGCS基因含有15个外显子和14个内含子,编码492个氨基酸.该基因与拟南芥的GCS基因相比较,编码区域同源性较高,而启动子区域的序列没有显著的相似性.通过RT-PCR的方法确定OsGCS基因的转录起始位点可能位于翻译起始位点(ATG)上游211bp处.在L395突变体中,T-DNA是单拷贝形式插入在OsGCS基因的第二内含子和外显子连接处,并且造成了3个碱基的缺失.在重金属耐受性、OsGCS基因表达以及体内GsH含量方面突变体L395和对照中花11之间没有明显的差别.     

三角帆蚌GPX基因结构特征及抗性相关SNP的筛选

根据三角帆蚌(Hyriopsis cumingii)谷胱甘肽过氧化物酶(Glutathione peroxidase,GPX)基因cDNA序列,通过PCR和基因组步移法,从三角帆蚌基因组DNA中扩增出GPX基因全长及其5′调控区。序列分析表明,该基因序列全长6 708 bp,含有2个外显子,1个内含子。5′调控区为992 bp,含有启动子的核心序列TATA盒以及其他一些转录调控元件,如AP1、C/EBP、CdxA。2个外显子长度分别为273 bp和991 bp,内含子长度为4 491 bp。通过直接测序法在三角帆蚌抗性群体和易感群体中筛选GPX基因的SNPs,并研究这些多态性位点与抗逆性状的相关性。共获得了16个SNP位点,其中启动子区的A-99G位点、A-86C位点、A-49C位点,内含子区的A2841T位点、C2847T位点、G3146C位点、A3150G位点以及G4645T位点共8个SNP位点的基因型频率和等位基因频率在抗性和易感群体中均存在显著性差异(P<0.05)。连锁不平衡分析结果显示GPX基因A-86C位点、A-49C位点、C2847T位点、A3150G位点与G4645T位点之间,以及A2841T位点与G3146C位点之间均存在强连锁不平衡。同时单倍型分析发现,在抗性群体中单倍型分别为ACTGT和TG的个体出现的频率显著高于易感群体中出现的频率。这些结果表明,GPX基因的部分SNPs可作为三角帆蚌抗病辅助育种的候选分子标记。     

水稻OsBP-73基因表达需要其内含子参与

该实验室以前的研究表明,水稻OsBP-73基因含有2个外显子和1个长度为2 471 bp的内含子.该文报告用OsBP-73基因ATG翻译起始密码子(在第1外显子中)上游序列(1- 818～ 215)与GUS基因构成嵌合质粒pRSSl,将该质粒转化水稻后,在抗性愈伤组织和转基因植株中未能检测到GUS基因的表达.只有用含有完整的内含子及其上游序列(1 818～ 2 844)与GUS基因构成嵌合质粒(p13GNF)时,才能在p13GNF的转基因抗性愈伤组织和植株中检测到GUS基因的表达.实验还证明,单是内含子序列并不能驱动GUS基因在转基因水稻中表达.由此推测:OsBP-73基因的启动子序列驱动基因表达时,需要基因内含子的参与.     

东方蜜蜂气味受体基因Or1和Or2的克隆与序列分析

本研究采用自行设计的引物对东方蜜蜂Apis cerana Fabricius气味受体(odorant receptors)Or1、Or2的部分基因组序列(GenBank登录号为:JN544932,JN544931)进行了克隆、测序和分析,以探寻传统气味受体(AcOr1)和非典型气味受体(AcOr2)基因在近缘种昆虫间的进化差异。试验所得的东方蜜蜂气味受体基因Or1、Or2的序列长度分别为1247bp和1138bp,各包含4个和2个内含子,编码区序列长度分别为682、686 bp。经序列比对发现,两气味受体DNA序列在东、西方蜜蜂及熊蜂间差异较大,最低相似性仅为56%(AcOr1—BtOr82a-like),差异的主要来源为内含子长度及其碱基的变异,而编码区氨基酸序列相似性较高,均达85%以上;从整体分析来看,在膜翅目昆虫中,非典型气味受体AcOr2较传统气味受体AcOr1是相对保守的气味受体基因。     

中国汉族个体HLA-A、-B基因全长序列的测定及调控区多态性

文章利用20个中国汉族个体样本建立了稳定精确的HLA-A、-B基因全长序列的克隆测序方法, 获得HLA-A 10个等位基因4.2 kb序列, HLA-B 6个等位基因3.7 kb序列, 序列涵盖了两个基因的所有外显子、所有内含子、5′启动子区以及3′非翻译区(3′UTR)。A*1153是文章发现的一个新等位基因, B*151101的内含子序列、5个HLA-A以及2个HLA-B等位基因的5′启动子序列和3′UTR序列为国际上首次报道, 其他等位基因均延伸了IMGT/HLA数据库中释放的全长序列。文章首次在中国汉族个体中测定了IMGT/HLA数据库中没有覆盖的HLA-A、-B基因的上游5′启动子以及下游3′UTR区域的多态性模式。HLA-A基因5′启动子延伸区域共发现26个SNPs和一处3 bp(AAA/-)的插入/缺失, 3′UTR延伸区域共发现14个SNPs; HLA-B基因5′启动子延伸区域共发现5个SNPs和一处1 bp(T/-)的插入/缺失, 3′UTR延伸区域共发现8个SNPs。通过对两个基因的5′启动子、外显子以及3′UTR的系统发育树分析, 发现两个基因调控区与外显子的进化关系有所不同, HLA-A基因除A*24020101外, 其他等位基因两端调控区与外显子连锁比较紧密, HLA-B基因两端调控区与外显子之间则发生了较为频繁的重组事件。     

家蚕谷胱甘肽-S-转移酶基因BmGSTz1的克隆、序列分析及组织表达特征

谷胱甘肽-S-转移酶(GSTs)是一个功能广泛的超基因家族, 其中Zeta家族在动物、植物和细菌中均有分布。在哺乳动物中, Zeta GSTs具有马来酰乙酰乙酸异构酶（MAAI）活性, 参与苯丙氨酸/酪氨酸的代谢过程。本研究对家蚕Bombyx mori基因组中预测的GST基因（BmGSTz1）进行了表达序列标签的搜索, 经拼接后获得一条含有3′和5′非翻译区在内的长度为1 239 bp 的cDNA序列, 其3′端含有AATAAA加尾信号。BmGSTz1基因含有4个内含子, 外显子/内含子边界均符合GT-AG 规则。经TA克隆证实, BmGSTz1基因编码区序列全长648 bp, 共编码215个氨基酸。BmGSTz1推定的分子量为24.8 kD, 等电点pI为8.06。BmGSTz1与其他昆虫和哺乳动物GSTz1的氨基酸序列高度保守, 进化分析表明家蚕BmGSTz1与黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae、意大利蜜蜂Apis mellifera和赤拟谷盗Tribolium castaneum的GSTz1形成1∶1∶1∶1∶1的直系同源关系。RT-PCR和基因芯片数据表明BmGSTz1在家蚕5龄第3天幼虫各组织中都有表达。序列和组织表达特征分析结果提示家蚕BmGSTz1可能具有MAAI活性, 这将为进一步深入研究BmGSTz1基因的功能提供参考。     

Study on the Relationship Between TSHR Gene and Thyroid Diseases

Thyroid stimulating hormone receptor (TSHR) is thought to play a critical role in the pathogenesis of certain thyroid diseases, including Graves' disease (GD), multinodular thyroid goiter (MTG), and Hashimoto's thyroiditis (HT). In order to understand whether single nucleotide polymorphisms in the TSHR gene contribute to thyroid diseases, we have conducted a case-control study in which, we examined 8 TSHR gene single-nucleotide polymorphisms in introns 1, 4, 5, 6 and exons 7 and 8, respectively, among patients with thyroid diseases. These included one family with GD (3 patients and 9 healthy members); 60 patients with familiar thyroid diseases (30 with GD, 20 with MTG, and 10 with HT patients), 48 sporadic patients with GD and 96 healthy control individuals. Direct sequencing of all 10 exons and part of introns of TSHR gene, in these patients as well as healthy controls revealed eight polymorphisms. A novel polymorphism in exon 8 AGA(Arg) → CGA(Arg). However, there were no significant differences between patients and controls in the incidence of these polymorphisms. These results suggest that the polymorphisms (polymorphism in intron 1 at 81 bp upstream of exon 2; polymorphism in intron 4 at 135 bp upstream of exon 5; polymorphism in intron 4 at 365 bp upstream of exon 5; polymorphism in intron 5 at 69 bp upstream of exon 6; means polymorphism in intron 6 at 13 bp downstream of exon 6; polymorphism in intron 6 at 187 bp upstream of exon 7; E7+16: polymorphism in 16 bp of exon 7; polymorphism in 40 bp of exon 8) of the TSHR gene may not contribute to the pathogenesis of thyroid diseases.     

Molecular cloning of cDNAs and genes for three alpha-glucosidases from European honeybees, Apis mellifera L., and heterologous production of recombinant enzymes in Pichia pastoris

cDNAs encoding three alpha-glucosidases (HBGases I, II, and III) from European honeybees, Apis mellifera, were cloned and sequenced, two of which were expressed in Pichia pastoris. The cDNAs for HBGases I, II, and III were 1,986, 1,910, and 1,915 bp in length, and included ORFs of 1,767, 1,743, and 1,704 bp encoding polypeptides comprised of 588, 580, and 567 amino acid residues, respectively. The deduced proteins of HBGases I, II, and III contained 18, 14, and 8 putative N-linked glycosylation sites, respectively, but at least 2 sites in HBGase II were unmodified by N-linked oligosaccharide. In spite of remarkable differences in the substrate specificities of the three HBGases, high homologies (38-44% identity) were found in the deduced amino acid sequences. In addition, three genomic DNAs, of 13,325, 2,759, and 27,643 bp, encoding HBGases I, II, and III, respectively, were isolated from honeybees, and the sequences were analyzed. The gene of HBGase I was found to be composed of 8 exons and 7 introns. The gene of HBGase II was not divided by intron. The gene of HBGase III was confirmed to be made up of 9 exons and 8 introns, and to be located in the region upstream the gene of HBGase I.     

Characterization of the rat carbonic anhydrase II gene structure: sequence analysis of the 5′ flanking region and 3′ UTR

The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5′ flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific β-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3′ UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.     

奶牛产奶性状与乳房炎相关基因CpG含量及分布特征的比较分析

DNA甲基化是表观遗传学的重要组成部分,基因启动子区及第一外显子区的CpG甲基化通常抑制该基因的表达,而去甲基化则促进基因表达。已有的研究发现荷斯坦牛的乳房炎指标SCC(Somatic cell count)与产奶量呈较强负相关。文章分析并比较了这两类性状的相关基因的启动子区、第一外显子、下游2 000 bp序列中CpG含量及分布特征。结果表明,乳房炎相关基因的启动子、第一外显子中CpG含量显著低于产奶性状相关基因,而两类性状基因下游2 000 bp序列中CpG含量无显著性差异。另外,文中提出了两个量化基因序列中CpG特征的指标,一个是CpG平均距离,用来衡量序列中的CpG分布;另一个是条件概率p(G|C),用以量化序列中二核苷酸CpG随碱基C出现的可能性,并对两类基因的启动子和第一外显子区域的这两个指标做了统计检验。研究结果对产奶性状与乳房炎相关基因的DNA甲基化调控研究奠定了基础。     

Structure of the murine complement factor H gene

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.