鲤sPLA2-III基因结构、系统发育和表达特征

磷脂酶A₂在磷脂代谢、炎症反应、细胞增殖和动脉粥样硬化等生理病理过程中发挥作用。为了探究sPLA₂-III在鲤(Cyprinus carpio Linnaeus)中的生物学功能, 实验使用BLAST同源搜索和线性分析在鲤 (Cyprinus carpio)基因组中挖掘得到5个sPLA₂-III基因, 分别位于5个连锁群, 分别命名为Ccpla2g3a1、Ccpla2g3a2、Ccpla2g3b、Ccpla2g3c1和Ccpla2g3c2。基因结构和序列分析显示Ccpla2g3as含有7个外显子, Ccpla2g3b和Ccpla2g3cs则均含有4个外显子, 分别编码530、525、461、752和753个氨基酸, 均含有PLA₂_bee_venom_like region和sPLA₂-III特征序列。线性分析显示, Ccpla2g3a1和Ccpla2g3a2, Ccpla2g3c1和Ccpla2g3c2来自鲤特异的染色体加倍事件。从2R (Two rounds of genome duplication)鱼雀鳝 (Lepisosteus oculatus)到3R(Fish-specific genome duplication, FSGD)鱼斑马鱼(Danio rerio)等, 雀鳝的pla2g3a(b)加倍为3R鱼的pla2g3a和pla2g3b, pla2g3c因在3R鱼的一个连锁群上丢失, 故3R鱼中只保留一个基因。同源性分析表明已有鱼类Pla2g3a、Pla2g3b和Pla2g3c垂直同源蛋白之间相似性分别为48.8%—93.2%、37.6%—74.3%和49.6%—97.6%, 鲤和斑马鱼的垂直同源基因之间相似性最高。系统发育结果显示, 鱼类及其祖先雀鳝的Pla2g3c以100%的置信值归在一支, 雀鳝Pla2g3a(b)与除了鲤科鱼类 (鲤和斑马鱼) Pla2g3b外的Pla2g3a和Pla2g3b以96%置信值在一支, 鲤和斑马鱼Pla2g3b单独形成一支表明在进化过程中该基因变异较大。荧光定量PCR结果表明Ccpla2g3as在整个鲤早期发育阶段表达量均较低, 在成鱼肝脏中表达量最高 (P<0.01); Ccpla2g3b在鲤受精后0.5h的表达量显著高于之后各取样点 (P<0.05), 在成鱼卵巢中表达量最高 (P<0.01); Ccpla2g3cs在120h表达量达到最高, 在成鱼脑中表达量最高 (P<0.01)。实验比较全面地揭示了鲤sPLA₂-III亚家族基因结构、系统发育和表达特征。     

建鲤肠型脂肪酸结合蛋白基因的分离及其SNPs与增重的相关分析

文章采用PCR方法从建鲤 (Cyprinus carpio var. jian) 基因组分离到2个肠型脂肪酸结合蛋白基因 (Fatty acid binding protein 2, FABP2), ORF长度均为399 bp, 相似度为92.2%, 分别记为jlFABP2a、jlFABP2b, 和斑马鱼 (Danio rerio) FABP2 ORF的相似度分别为88.0%和90.5%。jlFABP2s基因结构与FABPs家族其他成员一致, 由4个外显子和3个内含子组成, 2a和2b间内含子序列和长度差异明显。系统树显示这2个基因对应斑马鱼的1个FABP2基因, 和鲤鱼染色体数是斑马鱼的2倍一致。实时荧光定量PCR结果显示, jlFABP2a、2b在建鲤肠中的表达量极显著高于脑、肝脏、肌肉、肾脏、心脏、性腺等其他组织 (P<0.01), 且2a表达量显著 (雄鱼, P<0.05) 或极显著 (雌鱼, P<0.01) 高于2b, 但在其他组织则2b表达量稍高, 暗示2a为肠特异性表达, 2b则为广谱表达。通过比对8尾建鲤的2a和2b基因序列, 在2a和2b上分别找到12个和4个SNP, 均位于内含子上。使用PCR-RFLP 法检测jlFABP2a 上4个SNP 位点I1-A15G、I1-A99G、I2-C487T和I3-A27T在建鲤选育群体中的基因型分布, 并进行了基因型与个体增重的关联分析, 结果表明, 4个位点与雌、雄成鱼阶段增重分别有极显著或显著相关。同时考虑4个位点的基因型与增重的关系, 结果基因型AGGGCCXX 和AGGGXXAT的个体平均增重比其他个体快15%, 这两种基因型个体在选育群中占了9%, 具有较大的选育空间, 可用于建鲤分子育种计划中。     

鲤sPLA_2-Ⅲ基因结构、系统发育和表达特征

磷脂酶A_2在磷脂代谢、炎症反应、细胞增殖和动脉粥样硬化等生理病理过程中发挥作用。为了探究sPLA_2-Ⅲ在鲤(Cyprinus carpioLinnaeus)中的生物学功能,实验使用BLAST同源搜索和线性分析在鲤(Cyprinus carpio)基因组中挖掘得到5个sPLA_2-Ⅲ基因,分别位于5个连锁群,分别命名为Ccpla2g3a1、Ccpla2g3a2、Ccpla2g3b、Ccpla2g3c1和Ccpla2g3c2。基因结构和序列分析显示Ccpla2g3as含有7个外显子,Ccpla2g3b和Ccpla2g3cs则均含有4个外显子,分别编码530、525、461、752和753个氨基酸,均含有PLA_2_bee_venom_like region和sPLA_2-Ⅲ特征序列。线性分析显示, Ccpla2g3a1和Ccpla2g3a2, Ccpla2g3c1和Ccpla2g3c2来自鲤特异的染色体加倍事件。从2R (Two rounds of genome duplication)鱼雀鳝(Lepisosteus oculatus)到3R(Fish-specific genome duplication, FSGD)鱼斑马鱼(Danio rerio)等,雀鳝的pla2g3a(b)加倍为3R鱼的pla2g3a和pla2g3b, pla2g3c因在3R鱼的一个连锁群上丢失,故3R鱼中只保留一个基因。同源性分析表明已有鱼类Pla2g3a、Pla2g3b和Pla2g3c垂直同源蛋白之间相似性分别为48.8%—93.2%、37.6%—74.3%和49.6%—97.6%,鲤和斑马鱼的垂直同源基因之间相似性最高。系统发育结果显示,鱼类及其祖先雀鳝的Pla2g3c以100%的置信值归在一支,雀鳝Pla2g3a(b)与除了鲤科鱼类(鲤和斑马鱼) Pla2g3b外的Pla2g3a和Pla2g3b以96%置信值在一支,鲤和斑马鱼Pla2g3b单独形成一支表明在进化过程中该基因变异较大。荧光定量PCR结果表明Ccpla2g3as在整个鲤早期发育阶段表达量均较低,在成鱼肝脏中表达量最高(P0.01);Ccpla2g3b在鲤受精后0.5h的表达量显著高于之后各取样点(P0.05),在成鱼卵巢中表达量最高(P0.01);Ccpla2g3cs在120h表达量达到最高,在成鱼脑中表达量最高(P0.01)。实验比较全面地揭示了鲤sPLA_2-Ⅲ亚家族基因结构、系统发育和表达特征。     

建鲤GHR基因多态性及与增重相关的SNP位点的筛选

生长激素是调控动物生长的关键因子,它通过与膜蛋白生长激素受体结合后发挥作用,因此生长激素受体基因的变异对动物生长有着重要的影响。实验根据已分离的4个建鲤jlGHRs基因,通过测定来自6尾建鲤的序列,共找到38个SNP位点。使用PCR-RFLP方法检测了353尾建鲤在其中5个SNP位点(1a内含子3 A43G、1a外显子8 A361G、1b外显子8 C12T、2a外显子8 A555G和2b外显子8 A315G)的基因型,分析了不同基因型与生长性状的相关性。结果表明5个位点均与增重显性相关;1a内含子3 A43G还与体高/体长和体厚/体长显性相关;1b外显子8 C12T与体高/体长和尾柄高/尾柄长显性相关;1a外显子8 A361G和2a外显子8 A555G也与尾柄高/尾柄长显性相关。数据分析还显示增重标记个数富集4个以上的个体明显比标记少的个体生长快,在选育群中增重标记数有2个的个体最多占30%,而增重标记数4个以上的只占14%,说明存在着较大的选育空间。实验筛选的5个位点可以作为建鲤分子育种的有效标记。       

水稻中一个人类肿瘤抑制基因同源物的克隆与表达分析(英)

用同源筛选方法 ,从水稻 (OryzasativaL .)基因组文库中分离到一个与人类肿瘤抑制基因QM具有同源性的基因 ,命名为OSQM1。该基因包括 4个外显子和 3个内含子 ,编码 2 19个氨基酸 ,其中有 4 6个碱性氨基酸 ,其等电点高达 11.0 2。同源性搜寻发现此基因存在于真核生物中而且保守性较强 ,表明它可能具有重要的作用。North ern分析结果表明 ,它在不同的水稻器官中都有表达 ,但在花和愈伤组织中的表达水平明显低于其他营养器官。它在根和叶中的表达水平受环境因素的影响。     

异源四倍体鲫鲤及其原始父母本细胞周期蛋白B基因克隆与分子遗传分析

细胞周期蛋白(cyclin)B是真核细胞周期运转中调控G2期至M期转化的关键因子.本实验根据斑马鱼细胞周期蛋白 B1基因的剪切方式,设计3对特异于金鱼和银鲫细胞周期蛋白B基因外显子区兼并引物,首次扩增出异源四倍体鲫鲤及其原始亲本红鲫和鲤鱼细胞周期蛋白B基因2条大小分别约为2.4 kb和2.1 kb的片段.测序及比对分析表明：这3种鱼的细胞周期蛋白B基因2个片段均包含8个外显子和7个内含子.内含子剪切位点符合GT/AG规则,推测细胞周期蛋白B基因2个片段可能是细胞周期蛋白B基因的2种存在形式.异源四倍体鲫鲤与其原始父母本细胞周期蛋白B基因片段序列的比较结果表明：无论是外显子区还是内含子区,异源四倍体鲫鲤与其原始亲本都具有较高的遗传相似性,在1 025 bp的外显子序列中相同的碱基位点数达963个,为异源四倍体鲫鲤来源于红鲫和鲤鱼提供了分子证据;同时,异源四倍体鲫鲤与其原始亲本差异碱基位点的存在又表明这一独特的多倍体物种与其原始亲本存在着进化上的变异.此外,还分别以细胞周期蛋白B基因外显子和内含子序列构建了包括异源四倍体鲫鲤及其原始父母本在内的系统进化树.结果初步表明：对于亲缘关系较近的物种,用外显子和内含子序列构建的系统进化树与传统的物种进化树一致;而对于亲缘关系较远的物种,用内含子序列构建的进化树与传统的物种进化顺序存在较大差异.     

水稻中一个人类肿瘤抑制基因同源物的克隆与表达分析

用同源筛选方法,从水稻 (Oryza sativa L.) 基因组文库中分离到一个与人类肿瘤抑制基因QM具有同源性的基因,命名为OSQM1.该基因包括4个外显子和3个内含子,编码219个氨基酸,其中有46个碱性氨基酸,其等电点高达11.02.同源性搜寻发现此基因存在于真核生物中而且保守性较强,表明它可能具有重要的作用.Northern分析结果表明,它在不同的水稻器官中都有表达,但在花和愈伤组织中的表达水平明显低于其他营养器官.它在根和叶中的表达水平受环境因素的影响.     

水稻中一个人类肿瘤抑制基因同源物的克隆与表达分析

用同源筛选方法,从水稻（Oryza sativa L.）基因组库中分离到一个与人类肿瘤抑制基因QM具有同源性的基因,命名为OSQM1,该基因包括4个外显子和3个内含子,编码219个氨基酸,其中有46个碱性氨基酸,其等电点高达11.02。同源性搜寻发现此基因存在于真核生物中保守性较强,表明它可能具有重要的作用,North-ern分析结果表明,它在不同的水稻器官中都有表达,但在花和愈伤组织中的表达水平明显低于其他营养器官,它在根和叶中的表达水平受环境因素的影响。     

荠菜LEAFY同源基因的克隆与进化分析

LEAFY同源基因是高等植物花的分生组织分化的重要调节基因。根据已发表的LEAFY同源基因序列保守区设计引物,以荠菜(Capsellabursa-pastoris(L.)Medic.)基因组DNA序列为模板,克隆了一条长2866bp的LEAFY同源基因。序列分析表明,该基因含有3个外显子和2个内含子,外显子编码424个氨基酸组成的多肽。其单个外显子核苷酸序列与拟南芥(Arabidopsisthaliana)LEAFY基因同源性在90%以上,氨基酸序列同源性为86%,而与琴叶拟南芥(Ara-bidopsislyrata)的氨基酸序列同源性高达90%。不同植物物种的LEAFY同源氨基酸序列在C端高度保守,而N端则有较大程度的变异。3个外显子进化速率不同可能是由于所受选择压力不同所致。存在于荠菜CapLFY基因346位上的精氨酸突变可能是造成荠菜两种生态型花期不同的原因。     

人同源框基因Lhx4 cDNA序列的获得,染色体定位和基因组分析

Lhx4基因是LIM同源框基因家族的一员 ,它在运动神经元的发育过程中发挥着重要的作用 .从人脊髓cDNA文库中筛选到了 1个人源性Lhx4基因的cDNA全长序列 ,它与鼠源性的Lhx4基因的cDNA序列有 92 %同源性 .它的基因被定位在 1号染色体 1q 2 4 .1- 1q 2 4 .3的位置 ,并包含有 6个外显子 .其中同源框结构域由外显子 4和 5表达 ,LIM结构域 1由外显子 2表达 ,LIM结构域2由外显子 3表达 .     

大熊猫生长激素受体(GHR) cDNA 的克隆与序列分析

根据已报道的若干物种GHR 基因cDNA 序列设计引物, 利用RT- PCR 技术首次从大熊猫肝脏组织总RNA中扩增出GHR 基因编码区全长cDNA 序列, 克隆于pGEM®-T 载体后进行测序和序列分析。结果表明,大熊猫GHR 的ORF为1 917 bp , 编码638 个氨基酸的前体蛋白, 由18 个氨基酸的信号肽和620 个氨基酸的成熟肽组成,与人、狗、猪GHR 结构相似, 大熊猫GHR 成熟肽由246 个氨基酸的胞外区、24 个氨基酸的跨膜区和350 个氨基酸的胞内区组成, 并具GHR 的特征性结构。序列相似性比较显示, 大熊猫GHR 与哺乳类GHR 具有69 %～93 %的高序列相似性, 与爬行类和鸟类的序列相似性也达到60 % , 而与鱼类的序列相似性较低, 仅为30 %左右。与其它哺乳动物GHR 相比, 大熊猫GHR 在氨基酸序列上也存在明显的特异性。     

Isolation, characterization, and distribution of two cDNAs encoding for growth hormone receptor in rainbow trout (Oncorhynchus mykiss)

Growth hormone (GH) plays important roles in a vast array of physiological processes, including growth, metabolism, and reproduction. In this study, cDNAs for two unique growth hormone receptor variants were cloned and sequenced from rainbow trout. The two cDNAs, one consisting of 2920 bp and the other of 2820 bp, share 87.2% identity in nucleotide sequence and 85.5% identity in deduced amino acid sequence and presumably arose through gene duplication. The cDNAs encode for putative 593- and 594-amino acid growth hormone receptors (designated GHR1 and GHR2, respectively), each containing a single transmembrane domain and other motifs characteristic of the receptor family. Both GHR1 and GHR2 mRNAs were present in all tissues examined. Trout GHR mRNAs are differentially expressed, both in terms of abundance among tissues and in terms of abundance within selected tissues. GHR1 was more abundant than GHR2 in the brain, whereas GHR2 was more abundant than GHR1 in pancreas and spleen. These findings expand our understanding of the evolution of the GH receptor family and suggest that independent mechanisms serve to regulate the tissue-specific expression of GHR mRNAs.     

Characterization of a novel growth hormone receptor-encoding cDNA in rainbow trout and regulation of its expression by nutritional state

To clarify the divergence of the growth hormone receptor (GHR) family, we characterized a novel GHR from a teleost fish (rainbow trout). A 2357-nt cDNA was isolated and found to contain a single initiation site 71 nt from the most 5′ end, an open reading frame of 1971 nt encoding a 657-amino acid protein, and a single polyadenylation site 229 nt from the poly-A tail. Based on structural analysis, the protein was identified as a type 1 GHR (GHR1). The new GHR1 shares 42% and 43% amino acid identity, respectively, with GHR2a and GHR2b, the two type 2 GHRs isolated from trout previously. GHR1 mRNA was found in a wide array of tissues with the highest expression in the liver, red muscle, and white muscle. Fasting animals for 4 weeks reduced steady state levels of GHR1 in the liver, adipose, and red muscle. These findings help clarify the divergence and nomenclature of GHRs and provide insight into the function of duplicated GHR types.     

版纳微型猪近交系 GHR 基因克隆及生物信息学分析简

目的 获得版纳微型猪近交系（BMI）生长激素受体基因（GHR）序列,通过生物信息学分析预测GHR功能并进行GHR mRNA多组织表达谱分析.方法 以版纳微型猪近交系的肝脏组织为材料提取RNA,RT-PCR方法扩增GHR基因编码区序列,将序列连接至pMD18-T载体进行克隆、测序和生物信息学分析;半定量PCR检测GHR mRNA在BMI不同组织中表达量的差异.结果克隆出了BMI GHR 编码区序列,提交GenBank获得登录号KC999114.该基因CDS长1917 bp,编码638个氨基酸.生物信息学分析表明,与长白猪的GHR序列相比BMI存在4处氨基酸替换,分别为p.E381D、p.A409S、p.L556V和p.A580G,均发生在胞内域.GHR基因多组织表达谱分析显示：GHR mRNA几乎在各组织中均有表达,在肌肉中表达量最高,在小肠、心、肝、神经纤维、脾、卵巢中表达量较高,在肺、胃、大脑、胰和肾中的表达量较低.结论 成功克隆了版纳微型猪近交系GHR全长编码区序列,进行了生物信息学功能分析和组织表达谱分析,为进一步阐明版纳微型猪近交系生长矮小机理奠定了基础.     

成年核移植山羊生长相关基因的表达分析

体细胞核移植过程有可能影响克隆动物生长相关基因尤其是印迹基因的表达水平。本研究运用同源引物 PCR 扩增、RACE 技术并结合同源克隆策略, 克隆了 7 个山羊生长相关基因包括 3 个印迹基因(H19、IGF2 和 IGF2R)和 4 个非印迹基因(IGF1、IGF1R、GHR 和 GHSR)的完全 CDS 或者部分 cDNA 序列, 经生物信息学技术确认后, 用荧光实时定量 PCR 对 8只成年克隆山羊中这些基因的表达水平进行分析, 结果表明 3 个印迹基因中 IGF2R 基因表达水平极显著高于对照组的自然繁殖山羊(P<0.01), 而 H19 和 IGF2 的表达则没有很大区别; 4 个非印迹基因中只有 IGF1R 的表达水平极显著高于对照组(P<0.01), IGF1、GHR 和 GHSR 的表达与对照组相似。表明即使在表型正常的成年克隆动物也存在一定的表观遗传异常。通过对获得完全 CDS 和 3′UTR 的 IGF2 基因经过生物信息学分析表明, 山羊 IGF2 基因包含一个 540 bp 的开放阅读框 (ORF)编码 179 个氨基酸。IGF2 基因 cDNA 序列和氨基酸序列以及其它基因部分序列比较分析表明, 山羊所有这些基因与绵羊的同源性要高于同牛的同源性。     

Determinants of growth hormone receptor down-regulation

GH receptor (GHR) is a cytokine receptor family member that responds to GH by activation of the receptor-associated tyrosine kinase, JAK2 (Janus family of tyrosine kinase 2). We previously showed that JAK2, in addition to being a signal transducer, dramatically increases the half-life of mature GHR, partly by preventing constitutive GHR down-regulation. Herein we explored GHR and JAK2 determinants for both constitutive and GH-induced GHR down-regulation, exploiting the previously characterized GHR- and JAK2-deficient gamma2A reconstitution system. We found that JAK2's ability to protect mature GHR from rapid degradation measured in the presence of the protein synthesis inhibitor, cycloheximide, depended on the presence of GHR's Box 1 element and the intact JAK2 FERM (band 4.1/Ezrin/Radixin/Moesin); domain, but not the kinase-like or kinase domains of JAK2. Thus, GHR-JAK2 association, but not JAK2 kinase activity, is required for JAK2 to inhibit constitutive GHR down-regulation and enhance GHR half-life. In cells that expressed JAK2, but not cells lacking JAK2, GH markedly enhanced GHR degradation. Like JAK2-induced protection from constitutive down-regulation, GH-induced GHR down-regulation required the GHR Box 1 element and an intact JAK2 FERM domain. However, a JAK2 mutant lacking the kinase-like and kinase domains did not mediate GH-induced GHR down-regulation. Likewise, a kinase-deficient JAK2 was insufficient for this purpose, indicating that kinase activity is required. Both lactacystin (a proteasome inhibitor) and chloroquine (a lysosome inhibitor) blocked GH-induced GHR loss. Interestingly, GH-induced GHR ubiquitination, like down-regulation, was prevented in cells expressing a kinase-deficient JAK2 protein. Further, a GHR mutant, of which all the cytoplasmic tyrosine residues were changed to phenylalanines, was resistant to GH-induced GHR ubiquitination and down-regulation. Collectively, our data suggest that determinants required for JAK2 to protect mature GHR from constitutive degradation differ from those that drive GH-induced GHR down-regulation. The latter requires GH-induced JAK2 activation and GHR tyrosine phosphorylation and is correlated to GHR ubiquitination in our reconstitution system.     

Trypsinogen-like cDNAs and quantitative analysis of mRNA levels from the Indianmeal moth, Plodia interpunctella

Two cDNA fragments encoding full-length trypsinogen-like proteins were cloned from larvae of two strains (RC688s and HD198r) of the Indianmeal moth, Plodia interpunctella (Hübner), which differed in their sensitivity to Bacillus thuringiensis protoxins. One cDNA fragment contained 874 nucleotides, including a 780-nucleotide open reading frame that encoded a trypsinogen-like protein (PiT2b). Another cDNA fragment amplified from both P. interpunctella strains contained 864 nucleotides including a 780 bp open reading frame encoding a second trypsinogen-like protein (PiT2c). The cDNA sequence of PiT2b shared 89% sequence identity with PiT2a, a trypsinogen-like protein cloned previously from this species. The cDNA sequences of PiT2a and PiT2c shared 83% identity. The cDNA sequence identity between PiT2b and PiT2c was 80%. The cDNA for PiT2b from strain RC688s was different at six nucleotide positions from that of PiT2b from strain HD198r. Five nucleotide replacements occurred in the open reading frame leading to amino acid changes at all five positions. There were five nucleotide differences in the cDNAs for PiT2c trypsinogen-like proteins from the two strains. Two nucleotide substitutions in the open reading frame resulted in replacements of two amino acid residues in the deduced protein sequences. Amino acid sequences for PiT2a and PiT2b shared 84% identity, but only 50% identity was observed between PiT2c and the other two trypsinogen-like proteins. The deduced amino acid sequences for PiT2b and PiT2c included both signal and zymogen activation peptides and amino acid sequence motifs which are conserved in seven homologous trypsinogen-like proteins from other insects. Typical features of the putative trypsinogen-like proteins from P. interpunctella included the serine proteinase active site triad (His(81), Asp(133), and Ser(233)), three pairs of cysteine residues for disulfide bridges, and three residues, Asp(227), Gly(250), and Gly(260), that help to confer trypsin-like specificity to the enzymes. Quantitative RT-PCR analyses showed that, in fourth instar larvae, RC688s had 1.6-fold higher PiT2a trypsinogen-like mRNA than did HD198r. Expression of PiT2b mRNA was 3.4-fold higher in HD198r than in RC688s. Expression of PiT2c mRNA was 2.8-fold higher in RC688s than in HD198r. Mean accumulation levels of mRNAs for all three trypsinogen-like proteins were slightly higher in RC688s than in HD198r based on total RNA, and 1.3-fold higher in RC688s than in HD198r based on wet weight of larval body tissues.