绵羊肌球蛋白轻链2基因的分子克隆与表达分析

肌球蛋白轻链2蛋白是哺乳动物肌球蛋白的重要成员之一。获得其基因序列,并对其特征和表达进行分析,可为进一步研究功能奠定基础。本研究以小尾寒羊背最长肌为试验材料,采用RACE等方法对绵羊肌球蛋白轻链2基因的cDNA序列进行克隆和测序、利用相关生物学软件对所得cDNA序列进行生物信息学预测、并利用qRT-PCR和Western印迹法对其在绵羊各种组织中的表达进行分析。结果获得该基因cDNA序列全长为776 bp,提交至GenBank中获得相应的登录号为KJ710702;该序列中的498 bp的开放读码框编码含有166个氨基酸残基的蛋白质。预测发现该蛋白质无信号肽和二硫键,但存在N-糖基化和磷酸化位点;二级结构中以α-螺旋为主;蛋白质序列比较发现绵羊MYL2与小鼠、人、大鼠、猪、牛等哺乳动物的同源性均在95%以上。mRNA和蛋白质表达谱均显示该基因在绵羊心肌中表达量最高,其次为背最长肌。     

Bax基因cDNA的分子克隆研究

细胞编程死亡（ｐｒｏｇｒａｍｅｄｃｅｌｌｄｅａｔｈ）是细胞生理性死亡的一种主要形式。Ｂａｘ具有抑制细胞编程死亡的作用。本研究采用ＰＣＲ方法,从人肿瘤细胞ＨＬ—６０ｃＤＮＡ文库中扩增出若干个ｂａｘｃＤＮＡ片段,然后将它们分别与ＰＧＥＭ—Ｔ测序质粒载体连接,并转化到大肠杆菌ＪＭ１０９中去。用蓝／白法筛选重组菌落,经酶切分析及ＰＣＲ鉴定,获得了插入片段大小约为０．４ｋｂ及１．１ｋｂ的ＢａｘｃＤＮＡ重组质粒ＰＢａｘｌ和ｐＢａｘ２。这些片段的测序及表达工作正在进行之中。     

绵羊cdh1基因cDNA全编码区的克隆及序列分析

在哺乳动物成体睾丸中,精子发生的过程开始于未分化的A型精原细胞的干细胞群.目前已有报道在小鼠未分化的A型精原细胞中特异性表达钙依赖性跨膜黏着蛋白基因(cdh1),但绵羊的cdh1基因全序列未见报道.为了更好地研究绵羊精原干细胞的特性,根据已报道的其他物种的cdh1基因的cDNA保守区设计引物,从成年蒙古绵羊睾丸中提取总RNA,采用RT-PCR和分子克隆方法克隆了蒙古绵羊cdh1基因cDNA全编码区.DNA序列测定结果与牛的核苷酸序列比对,同源性为96.5％,说明该基因在进化上是高度保守的.这为制备绵羊CDHI的抗体奠定了基础,并且为绵羊精原干细胞的分子水平鉴定提供了研究备件.     

牛aS1-酪蛋白基因5’-调节区的分子克隆

用牛αsl－酪蛋白cDNA作探针,从牛基因组文库中筛选出一段αsl－酪蛋白基因序列。由限制内切酶图谱和souther印迹分析可以确定其中含有完整的αs1-酪蛋白基因的5’调节区。     

蒙古绵羊 tnp1基因cDNA全编码区的克隆及序列分析

过渡蛋白1基因(tnp1)是圆形精子细胞特异表达的基因.绵羊tnp1基因的DNA序列至今尚未报道.为了开展绵羊圆形精子细胞标记基因的研究,根据其他物种tnp1基因cDNA的保守序列设计引物,从成年蒙古绵羊睾丸中提取总RNA,采用RT-PCR和分子克隆方法,克隆了蒙古绵羊tnp1基因cDNA全编码区.该基因cDNA 长246 bp,包含一个168 bp的ORF,编码含有54个氨基酸的多肽链.DNA序列测定结果与牛的核苷酸序列比对,同源性为94.0%.绵羊tnp1基因的cDNA克隆和序列测定为进一步研究绵羊精子发生过程奠定了基础.     

绵羊EGFR基因的克隆、表达及序列分析

表皮生长因子受体(epidermal growth factor receptor,EGFR)是酪氨酸激酶受体家族成员之一,不仅参与细胞增殖、生长和凋亡等多种生命活动,也可调节哺乳动物的乳腺发育及泌乳维持,但对绵羊EGFR基因的序列特征及组织表达情况鲜有报道.本试验以高泌乳量的小尾寒羊(泌乳高峰期和空怀期)及低泌乳量的甘肃高山细毛羊(泌乳高峰期)母羊为研究对象,利用RT-PCR、克隆及测序技术获得绵羊EGFR基因完整的CDS区,分析了 EGFR蛋白的结构特征及理化性质,利用RT-qPCR技术研究了基因的组织表达情况.结果表明,绵羊EGFR基因CDS区全长为3 627 bp,编码1 208个氨基酸.绵羊EGFR的氨基酸序列在各物种间较保守,与黄牛EGFR的氨基酸序列同源性最高.EGFR为跨膜蛋白,包含111个磷酸化位点,二级结构以α螺旋和无规则卷曲为主.网络互作分析表明EGFR蛋白与肝素结合表皮生长因子(HB-EGF)、表皮调节素(EREG)、双调蛋白(AREG)及生长因子受体结合蛋白2(GRB2)结合发挥作用.EGFR主要参与MAPK,PI3K/AKT,JAK/STAT及Wnt信号通路,从而参与了动物的乳腺发育及泌乳功能的调节.RT-qPCR结果表明,绵羊EGFR基因的表达具有组织特异性、时空特异性和品种特异性.该基因在所研究的8个组织中均表达,但在肾脏、卵巢、肝脏、乳腺和肺脏组织中的表达量较高;在小尾寒羊的乳腺组织中,该基因在空怀期的表达量显著高于泌乳高峰期的(P＜0.05);在泌乳高峰期的乳腺组织中,该基因在小尾寒羊中的表达量高于甘肃高山细毛羊的.本试验为深入研究绵羊EGFR基因的泌乳生物学功能提供了基础数据.     

小麦春化相关基因的分子克隆与功能分析

小麦春化相关基因的分子克隆与功能分析@种康$中国科学院植物研究所!北京100093@许智宏$中国科学院植物研究所!北京100093@谭克辉$中国科学院植物研究所!北京100093小麦;;基因;;分子克隆     

编码天麻抗真菌蛋白cDNA的分子克隆

用重组ＤＮＡ 技术研究了编码天麻抗真菌蛋白（ＧＡＦＰ）的基因,从天麻（Ｇａｓｔｒｏｄｉａ ｅｌａｔａＢｌ．）块茎中提取Ｐｏｌｙ（Ａ） ｍ ＲＮＡ 后合成ｃＤＮＡ,构建成表达型ｃＤＮＡ 文库,用纯化的蛋白质探针通过免疫筛选找出对应的ｃＤＮＡ 克隆。在进一步证明所选用的ｃＤＮＡ 克隆含有重组的λ－ｐｈａｇｅ ＤＮＡ 后,提取和纯化含有插入片段重组子的ＤＮＡ,用Ｅｃｏ ＲＩ酶切分析可见插入片段。已分离出编码天麻抗真菌蛋白的基因     

猪特异性脂肪细胞膜蛋白基因cDNA 5′端的分子克隆

猪特异性脂肪细胞膜蛋白是近年来才被报道的一种可能与前脂肪细胞生长、发育和脂肪沉积调控有关的蛋白质。采用PT-PCR和PACE技术,首次获得猪特异性脂肪细胞蛋白基因cDNA 5‘端的克隆和序列。     

Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle

The cDNA encoding a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147-residue polypeptide, which we termed CPI17, a 17-kDa PKC-potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC₅₀=0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non-muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.     

Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells

Phorbol ester induces actin cytoskeleton rearrangements in cultured vascular smooth muscle cells. Calponin and SM22 alpha are major components of differentiated smooth muscle and potential regulators of actin cytoskeleton interactions. Here we show that actin fibers decorated with h1 CaP remain stable, whereas SM22 alpha-decorated actin bundles undergo rapid reorganization into podosomes within 30 min of PDBu exposure. Ectopic expression of GFP alpha-actinin had no effect on the stability of the actin cytoskeleton and alpha-actinin was transported rapidly into PDBu-induced podosomes. Our results demonstrate the involvement of CaP and SM22 alpha in coordinating the balance between stabilization and dynamics of the actin cytoskeleton in mammalian smooth muscle. We provide evidence for the existence of two functionally distinct actin filament populations and introduce a molecular mechanism for the stabilization of the actin cytoskeleton by the unique actin-binding interface formed by calponin family-specific CLIK23 repeats.     

Molecular cloning and sequencing of the chicken smooth muscle myosin regulatory light chain

A cDNA probe was constructed from a chicken skeletal muscle regulatory light chain cDNA and was used to screen a chicken gizzard cDNA library. A clone containing the entire coding region of the chicken gizzard regulatory light chain was isolated and sequenced. The deduced protein sequence is identical to the most recently reported chemical sequence of the chicken smooth muscle regulatory light chain, and has homologies with other troponin C-like calcium-binding proteins.     

绵羊CCNG1基因克隆及表达分析

细胞周期蛋白cyclin G1(CCNG1)是一个重要的细胞周期调控因子,参与哺乳动物颗粒细胞增殖、卵母细胞成熟等繁殖生物学过程,但其在绵羊中鲜有报道。为了研究CCNG1基因对绵羊发情调控以及季节性繁殖的影响,文章对绵羊CCNG1基因进行了克隆和组织表达谱分析,利用Real-time PCR对该基因在多浪羊(常年发情)与中国美利奴羊(季节性繁殖)发情周期不同阶段性腺轴组织的表达变化进行了实时检测。获得了绵羊CCNG1基因部分cDNA序列,其中编码区全长885 bp,编码294个氨基酸。CCNG1蛋白结构经预测存在多个磷酸化位点和蛋白激酶C磷酸化位点。CCNG1基因在所检测绵羊各组织中均有表达,但在卵巢与肾脏中为高丰度表达;CCNG1在不同绵羊品种发情周期不同阶段性腺轴组织的表达变化规律基本一致,卵巢、子宫、松果体、垂体均是在发情期达到峰值。但是在发情期和发情后期卵巢CCNG1的表达量存在显著的品种间差异(P<0.01)。研究结果表明,CCNG1可能通过参与卵泡的生长发育继而达到对绵羊发情和季节性繁殖的调控。     

Molecular cloning of magnesium-independent type 2 phosphatidic acid phosphatases from airway smooth muscle.

Members of the type 2 phosphatidic acid phosphatase (PAP2) family catalyse the dephosphorylation of phosphatidic acid (PA), lysophosphatidate and sphingosine 1-phosphate. Here, we demonstrate the presence of a Mg(2+)-independent and N-ethymaleimide-insensitive PAP2 activity in cultured guinea-pig airway smooth muscle (ASM) cells. Two PAP2 cDNAs of 923 and 926 base pairs were identified and subsequently cloned from these cells. The ORF of the 923 base pair cDNA encoded a protein of 285 amino acids (Mr = 32.1 kDa), which had 94% homology with human PAP2a (hPAP2a) and which probably represents a guinea-pig specific PAP2a (gpPAP2a1). The ORF of the 926 base pair cDNA encoded a protein of 286 amino acids (Mr = 32.1 kDa) which had 84% and 91% homology with hPAP2a and gpPAP2a1, respectively. This protein, termed gpPAP2a2, has two regions (aa 21-33 and 51-74) of marked divergence and altered hydrophobicity compared with hPAP2a and gpPAP2a1. This occurs in the predicted first and second transmembrane domains and at the extremes of the first outer loop. Other significant differences between gpPAP2a1/2 and hPAP2a, hPAP2b and hPAP2c occur at the cytoplasmic C-terminal. Transient expression of gpPAP2a2 in Cos-7 cells resulted in an approx. 4-fold increase in Mg(2+)-independent PAP activity, thereby confirming that gpPAP2a2 is another catalytically active member of an extended PAP2 family.     

Molecular cloning and sequencing of the plasma-membrane Ca2+ pump of pig smooth muscle.

cDNAs coding for the plasma-membrane Ca2+ pump have been isolated from a pig smooth-muscle cDNA library and sequenced. The open reading frame encodes a protein of 1220 amino acids, which corresponds to the one already described in a human teratoma cell line. We demonstrate here that this cDNA probably represents the only isoform of the plasma-membrane Ca2(+)-transport ATPase expressed in this smooth muscle. There is no evidence for the expression of any other plasma-membrane Ca2(+)-pump gene, or for the presence of other alternatively spliced isoforms. These results are in apparent contradiction to those obtained on protein levels which demonstrate the reaction of at least two different polypeptides with a panel of antibodies against the plasma-membrane ATPase. It is suggested that these two polypeptides could result from a post-translational modification of one single enzyme.     

Molecular pharmacology of isoprostanes in vascular smooth muscle

Isoprostanes are marker of lipid peroxidation and are produced after free-radical attack of membrane lipids. In addition, they are biologically active and are essentially vaso- and broncho-constrictor. Their smooth muscle constrictor actions are closely linked to the activation of the thromboxane A(2) receptor, but also involve a distinct receptor not yet identified. The response of vascular smooth muscle to isoprostanes is subclass-specific (F-series versus E-series isoprostanes) and cell- and species-related. In this review, we will address the vascular actions of isoprostanes and their possible role in vascular physiology and pathophysiology.     

Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle

Summary Gap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels