草莓ξ-胡萝卜素脱氢酶基因ZDS的克隆及特征分析

用RT-PCR和RACE技术从草莓(Fragaria ananassa Duchesne)果实中克隆到ξ-胡萝卜素脱氧酶基因ZDS,命名为FaZDS,其cDNA全长2148 bp,具有1个1710 bp的完整开放阅读框(ORF),编码569个氨基酸.序列分析表明,FaZDS编码的氨基酸序列与其它植物的ZDS蛋白有很高的相似性.系统进化树分析表明,草莓与苹果的ZDS蛋白亲缘关系较近.原核表达结果表明FaZDS基因在大肠杆菌中能高效表达.用半定量RT-PCR技术进行组织表达模式分析表明,FaZDS基因在草每的花、叶片和果实中均有表达,表达量为花＞红果＞粉红果＞白果＞青果＞叶.     

草莓ζ-胡萝卜素脱氢酶基因ZDS的克隆及特征分析

用RT-PCR和RACE技术从草莓(Fragaria ananassa Duchesne)果实中克隆到ζ-胡萝卜素脱氢酶基因ZDS,命名为FaZDS,其cDNA全长2148 bp,具有1个1710 bp的完整开放阅读框(ORF),编码569个氨基酸。序列分析表明,FaZDS编码的氨基酸序列与其它植物的ZDS蛋白有很高的相似性。系统进化树分析表明,草莓与苹果的ZDS蛋白亲缘关系较近。原核表达结果表明FaZDS基因在大肠杆菌中能高效表达。用半定量RT-PCR技术进行组织表达模式分析表明,FaZDS基因在草莓的花、叶片和果实中均有表达,表达量为花红果粉红果白果青果叶。     

利用简并引物克隆黑莓、树莓肌动蛋白基因片段及表达分析

采用同源克隆法,利用设计的一对肌动蛋白(actin)基因简并引物,从黑莓、树莓和悬钩子杂种3个悬钩子植物品种均得到一条743 bp的actin cDNA片段,推测编码247个氨基酸.序列比对发现,克隆的actin基因推导的氨基酸序列和其他植物的肌动蛋白氨基酸序列具有高度保守性,经数据库搜索后将来自黑莓和树莓品种的actin cDNA片段在GenBank中登录,登录号分别为HQ439558和HQ439559.不同来源的植物actin蛋白系统进化树的聚类结果表明,树莓和悬钩子杂种亲缘关系较为密切.actin基因在3个悬钩子植物品种不同组织中的RT-PCR分析结果表明,其在检测的不同组织中均有一定程度的表达量,可能都属于组成型表达的肌动蛋白基因.研究结果为利用actin基因作为内参进一步研究黑莓和树莓其他基因的丰度奠定基础.     

棉花HSP70基因的克隆与原核表达

以抗旱性较强的棉花品种‘KK1543’为材料,采用RT-PCR技术克隆了1个棉花HSP70基因,命名为GhHSP70。研究结果表明:GhHSP70基因开放阅读框长1 997bp,编码648个氨基酸,GhHSP70蛋白相对分子量为70.94kD,等电点为4.83。氨基酸序列比对和系统进化树分析发现,GhHSP70与欧洲大叶杨HSP70的亲缘关系最近,氨基酸序列一致性达到96.4%。为进一步分析基因的功能,构建原核表达载体pGEX-4T-1-GhHSP70,并在大肠杆菌中异源表达。SDS-PAGE分析表明所表达蛋白与预期蛋白大小一致,重组蛋白在37℃,0.2mmol/L IPTG诱导2h时表达量最大。研究为进一步研究棉花HSP70基因功能奠定基础。     

柱花草WRKY转录因子在低磷胁迫下的克隆与分析

该研究依据生物信息学分析,采用RT-PCR方法从格拉姆柱花草[Stylosanthes guianensis (Aubl.)]中克隆出1个WRKY转录因子基因,命名为StWRKY45。该基因最大开放阅读框(ORF)为924bp,编码307个氨基酸,分子量为35.62kD,等电点为9.81。系统进化树分析表明,StWRKY45属于WRKY转录因子第Ⅱ类WRKY基因,与拟南芥AtWRKY45、AtWRKY57亲缘关系最近。实时荧光定量PCR结果表明,在非磷胁迫下(对照),StWRKY45基因在柱花草幼苗的根、茎、叶中都有表达,但表达量均相对较低;在低磷胁迫下,StWRKY45基因在格拉姆柱花草根、茎、叶中的表达基本随胁迫时间的延长逐渐升高,且均在叶中的表达量最高;在低磷胁迫96h时叶、茎中的相对表达量均达到最高,分别是对照的20.47倍、9.38倍,但在低磷胁迫72h时根中的相对表达量达到最高,为对照的9.29倍。研究表明,StWRKY45基因受低磷胁迫诱导高表达,推测StWRKY45基因可能参与柱花草对低磷胁迫的响应。     

中国水仙水杨酸甲酯合成酶基因克隆与表达分析

为了解中国水仙(Narcissus tazetta var. chinensis)花香气形成的分子机理,以花发育形成相关基因的SSH文库获得的cDNA片段为基础,利用RACE技术从中国水仙花朵中克隆了水杨酸甲酯合成酶基因,命名为NtSAMT1 (GeneBank No. JX273470),其cDNA全长1323 bp,包含1个1131 bp完整阅读框架,编码376个氨基酸,具有Methyltransf_7 superfamily蛋白保守区,与仙女扇(Clarkia breweri) SAMT蛋白(1m6eX)的三维结构相似。系统进化树分析表明,中国水仙与粳稻的SAMT蛋白亲缘关系最近。原核表达结果表明NtSAMT1在大肠杆菌中能高效表达。半定量RT-PCR分析表明,NtSAMT1在花蕾期就有表达,第1天完全开放时各部位均有表达,以雄蕊与雌蕊的表达量最高,第8天已检测不到表达。     

甘蔗类异黄酮还原酶(IRL)基因的克隆与表达分析

采用RT-PCR技术从甘蔗中克隆So IRL基因,用生物信息学方法对获得的氨基酸序列进行分析,利用荧光定量PCR技术研究So IRL基因在甘蔗不同组织和不同胁迫条件下的表达特性。结果表明,克隆获得甘蔗So IRL,Gen Bank登录号为KF808324。该c DNA全长1 169 bp,含有1个927 bp的完整开放阅读框(ORF),编码309个氨基酸。系统进化树分析显示,甘蔗So IRL与玉米的IRL蛋白亲缘关系较近。q RT-PCR分析表明So IRL在甘蔗根、茎、叶中均有表达;在RSD病菌及低温(4℃)、聚乙二醇(PEG)、Na Cl和脱落酸(ABA)4种非生物胁迫下均被诱导表达,但表达模式不同。说明该基因可能参与甘蔗应答RSD过程,并可能在非生物胁迫中也发挥了作用。     

小麦TaLSD1锌指蛋白基因的电子克隆及序列分析

采用电子克隆与RT-PCR相结合的技术,在条锈菌(Pucciniastriiformis f.sp.tritici)侵染的小麦中克隆了一个LSD1型锌指蛋白基因,命名为TaLSD1(GenBank登录号为EF553327)。序列分析表明,该基因全长1024bp,编码生成1个包含3个保守LSD1型锌指结构(CxxCxRxxLMYxxGASxVxCxxC)且长度为146个氨基酸的多肽。进化树分析表明,TaLSD1与水稻(Oryza sativa)、拟南芥(Arabidopsis thaliana)和芜菁(Brassica rapa)中部分含有3个保守LSD1型锌指结构的同源基因亲缘关系较近,而与其它包含不同数目的LSD1型锌指结构基因亲缘关系较远。推测TaLSD1在进化中丢失了部分序列,进而执行新的功能。半定量RT-PCR结果显示,该基因在亲和以及非亲和组合中的表达模式很相似,均表现在前期基因表达被抑制而后期恢复正常。初步推测TaLSD1在转录水平上的表达受光诱导,同时,作为一个细胞程序性死亡的负调控因子在小麦与条锈菌互作过程中起作用。     

广西麻鸡FOXL2基因的克隆、序列分析及在高低产蛋量卵巢中的表达

为了研究鸡FOXL2基因的结构和功能,本研究克隆了广西麻鸡FOXL2基因编码区序列,分析了其突变位点及与其他物种的同源性和进化距离,以及在高产组和低产组母鸡卵巢组织中的表达水平.结果 表明:广西麻鸡FOXL2基因的编码区长度为918 bp,共编码305个氨基酸,存在一处错义突变和两处同义突变.通过物种间的同源性分析显示,广西麻鸡FOXL2基因与原鸡(Gallus gallus)、雉鸡(Phasianus colchicus)、绿头鸭(Anas platyrhynchos)、野鸽(Columba livia)、人(Homo sapiens)、小鼠(Mus musculus)的同源性分别为99.7％、98.8％、95.1％、92.0％、75.7％、75.5％.通过种间进化树分析表明,广西麻鸡与原鸡(Gallus gallus)的亲缘关系最近,与小鼠(Mus musculus)的亲缘关系最远.FOXL2在高低产蛋量鸡卵巢中的表达水平结果显示:FOXL2基因在高产蛋组中的表达量显著高于低产蛋组中的表达量(P＜0.05).该研究结果提示鸡FOXL2的序列相对较保守,对卵巢功能的维持和提高产蛋量具有功能性作用.     

猪CuZnSOD基因的克隆、表达及功能分析

为了进一步了解和认识CuZnSOD基因的结构和功能,揭示CuZnSOD对猪抗氧化机能的影响,寻找与肉质性状相关联的分子标记,文章采用RACE(Rapid amplification of cDNA end)方法,对莱芜猪CuZnSOD基因cDNA进行克隆测序,分析其结构和功能,并用Real-timePCR检测CuZnSOD基因的表达.结果表明,CuZnSOD基因cDNA序列全长658 bp(GenBank登录号:GU944822),包含76 bp的5'UTR和120 bp的3'UTR序列.全部CDS序列462 bp,编码153个氨基酸,分子量为15.9 kDa,等电点为6.03.CuZnSOD基因编码的氨基酸序列中,第3氨基酸残基处存在1个O-糖基化位点,第86氨基酸残基处存在1个N-糖基化位点.二级结构中α螺旋仅占1.31%.在进化过程中高度保守,与人、牛、小鼠和褐鼠的编码区同源性分别为87.74%、87.66%、83.44%和83.23%;氨基酸序列同源性分别为90.26%、94.12%、92.21%和91.50%.CuZnSOD存在典型的金属结合配体结构域(GFHVHQFGDNT).基于蛋白序列所构建的分子进化树表明猪与牛的亲缘关系最近.在mRNA水平上,CuZnSOD是一个广谱表达基因,在大脑、心脏、脾脏、肝脏、肾脏、肺、大肠、小肠、脊髓,肌肉、背膘和胃中都能检测到,其在肾脏,小肠和肺中表达量较高,在心脏和肌肉组织中表达量较低.     

Coupling of osteopontin and its cell surface receptor CD44 to the cell survival response elicited by interleukin-3 or granulocyte-macrophage colony-stimulating factor

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) share a common beta subunit, the distal cytoplasmic domain of which is essential for the promotion of cell survival by these two cytokines. Genes whose expression is specifically induced by signaling through the distal cytoplasmic domain of this receptor beta subunit were screened by a subtraction cloning approach in derivatives of a mouse pro-B-cell line. One gene thus identified was shown to encode a protein highly homologous (with only 7 amino acid substitutions) to murine osteopontin (OPN), a secreted adhesion protein. Conditioned medium from cells expressing wild-type OPN, but not that from cells expressing a deletion mutant lacking residues 79 to 140, increased the viability of a non-OPN-producing cell line in the presence of human GM-CSF. Antibody blocking experiments revealed that OPN produced as a result of IL-3 or GM-CSF signaling was secreted into the medium and, through binding to its cell surface receptor, CD44, contributed to the survival-promoting activities of these two cytokines. Furthermore, coupling of the OPN-CD44 pathway to the survival response to IL-3 was also demonstrated in primary IL-3-dependent mouse bone marrow cells. These results thus show that induction of an extracellular adhesion protein and consequent activation of its cell surface receptor are important for the antiapoptotic activities of IL-3 and GM-CSF.     

Identification of Transglutaminase Reactive Residues in Human Osteopontin and Their Role in Polymerization

Osteopontin (OPN) is a highly posttranslationally modified protein present in several tissues where it is implicated in numerous physiological processes. OPN primarily exerts its functions through interaction with integrins via the Arg-Gly-Asp and Ser-Val-Val-Tyr-Gly-Leu-Arg sequences located in the N-terminal part of the protein. OPN can be polymerized by the cross-linking enzyme transglutaminase 2 (TG2), and polymerization has been shown to enhance the biological activity of OPN. However, little is known about the reactivity and location of the glutamine and lysine residues involved in the TG2-mediated modification of OPN. Here we show that TG2 catalyses the incorporation of 5-(Biotinamido)pentylamine at glutamines in both the N- and C-terminal parts of OPN, whereas TG2 primarily incorporated the glutamine-donor peptide biotinyl-TVQQEL-OH into the C-terminal part of OPN. By mass spectrometric analyses we identified Gln34, Gln42, Gln193 and Gln248 as the major TG2 reactive glutamines in OPN. The distribution of reactive Gln and Lys residues in OPN proved to be important, as the full-length protein but not the physiologically highly active integrin-binding N-terminal part of OPN were able to polymerize in a TG2-mediated reaction. Collectively, these data provide important new molecular knowledge about the mechanism of OPN polymerization.     

内蒙古白绒山羊IGF-IR基因cDNA克隆及组织表达特异性分析

旨在克隆内蒙古白绒山羊IGF-IR基因并分析其基本表达模式.采用RT-PCR克隆基因,将得到的IGF-IR基因cDNA片段的核苷酸序列及其编码的氨基酸序列进行生物信息学分析.半定量RT-PCR进行组织特异性表达检测.获得了内蒙古白绒山羊IGF-IR基因3’端编码区2118 bp的cDNA序列(JN200823),编码705个氨基酸残基.核苷酸序列与牛的IGF-IR( XM606794.3)基因同源性为98％,相应的氨基酸序列同源性为99％.SMART分析表明,推导出的编码蛋白具有跨膜域,酪氨酸激酶催化域.半定量RT-PCR检测表明,IGF-IR基因在绒山羊脑、胰腺、肝、肾组织中均有表达.     

大鼠动脉内膜损伤后血管中的骨桥蛋白和基质Gla蛋白mRNA的表达变化

本实验采用球囊剥脱大鼠主动脉内皮造成血管内膜损伤的芭Ｎｏｒｔｈｅｒｎ印迹分析,逆转录聚合酶链式反应（ＲＴ－ＰＣＲ）等方法,研究大鼠主动脉内损伤后血管中的骨桥蛋白（ｏｓｔｅｏｐｏｎｔｉｎ,ＯＰＮ）和基质Ｇｌａ蛋白（ｍａｔｒｉｘ Ｇｌａ ｐｒｏｔｅｉｎ,ＭＧＰ）ｍＲＮＡ水平的动态变化。与无内皮损伤血管比较,损伤后的血管中ＯＰＮ和ＭＧＰ的ｍＲＮＡ水平明显升高,损伤后１,７,１４ｄ,两者的ｍＲＮＡ水平逐渐     

骨桥蛋白通过NF-κB上调钙蛋白酶小亚基1促进肝癌细胞迁移

骨桥蛋白(osteopontin,OPN)参与调控多种信号途径激活转移相关基因,进而促进细胞迁移.钙蛋白酶小亚基1(calpain small subunit1,Capn4)与肿瘤转移密切相关,在许多肿瘤及其转移组织中高表达.为了探讨OPN促进肝癌细胞迁移的分子机制,应用报告基因检测、RT-PCR、免疫印迹及伤口愈合等方法检测了肝癌细胞中OPN对Capn4的调控作用及其对肝癌细胞迁移的影响.结果显示,在HepG2细胞中过表达OPN后,Capn4的启动子转录活性显著增强,同时mRNA及蛋白质表达水平也明显上调.在HepG2细胞中应用siRNA干扰OPN的表达可导致Capn4启动子转录活性受到明显抑制,同时mRNA及蛋白质表达水平也显著下调.应用核转录因子-κB(NF-κB)的抑制剂PDTC可抑制由过表达OPN导致的HepG2细胞中Capn4的上调.伤口愈合实验显示,OPN可以通过上调Capn4促进肝癌细胞迁移.因此,研究发现,OPN通过NF-κB上调Capn4的表达,进而促进肝癌细胞的迁移,这一发现对进一步阐明肝癌细胞迁移的分子机制具有重要意义.     

Osteopontin Is Induced by TGF-β2 and Regulates Metabolic Cell Activity in Cultured Human Optic Nerve Head Astrocytes

The aqueous humor (AH) component transforming growth factor (TGF)-β2 is strongly correlated to primary open-angle glaucoma (POAG), and was shown to up-regulate glaucoma-associated extracellular matrix (ECM) components, members of the ECM degradation system and heat shock proteins (HSP) in primary ocular cells. Here we present osteopontin (OPN) as a new TGF-β2 responsive factor in cultured human optic nerve head (ONH) astrocytes. Activation was initially demonstrated by Oligo GEArray microarray and confirmed by semiquantitative (sq) RT-PCR, realtime RT-PCR and western blot. Expressions of most prevalent OPN receptors CD44 and integrin receptor subunits αV, α4, α 5, α6, α9, β1, β3 and β5 by ONH astrocytes were shown by sqRT-PCR and immunofluorescence labeling. TGF-β2 treatment did not affect their expression levels. OPN did not regulate gene expression of described TGF-β2 targets shown by sqRT-PCR. In MTS-assays, OPN had a time- and dose-dependent stimulating effect on the metabolic activity of ONH astrocytes, whereas TGF-β2 significantly reduced metabolism. OPN signaling via CD44 mediated a repressive outcome on metabolic activity, whereas signaling via integrin receptors resulted in a pro-metabolic effect. In summary, our findings characterize OPN as a TGF-β2 responsive factor that is not involved in TGF-β2 mediated ECM and HSP modulation, but affects the metabolic activity of astrocytes. A potential involvement in a protective response to TGF-β2 triggered damage is indicated, but requires further investigation.     

The osteopontin-CD44 survival signal involves activation of the phosphatidylinositol 3-kinase/Akt signaling pathway

We have recently demonstrated that the gene encoding the osteopontin (OPN) protein is activated both by interleukin-3 and granulocyte-macrophage colony-stimulating factor signaling pathways and that, through binding to the cell surface receptor CD44, OPN contributes to the survival activities of interleukin (IL)-3 and GM-CSF (Lin, Y.-H., Huang, C.-J., Chao, J.-R., Chen, S.-T., Lee, S.-F., Yen, J. J.-Y., and Yang-Yen, H.-F. (2000) Mol. Cell. Biol. 20, 2734-2742). In this report, we demonstrate that the CD44-binding domain of OPN involves a region containing amino acid residues from 121 to 140 and that both threonine and serine at positions 137 and 147, respectively, are essential for the survival stimulatory effect of OPN. Substitution of either residue with alanine results into a dominant negative mutant that overrides the survival effect of IL-3. Upon binding to the CD44 receptor, the wild-type OPN but not the inactive mutant induces activation of phosphatidylinositol 3-kinase and Akt. Last, we demonstrate that two waves of Akt activation are detected in IL-3-treated cells and that the survival promoting effect of OPN is mediated predominantly through the phosphatidylinositol 3-kinase/Akt signaling pathway. Together, our results suggest that a positive autoregulatory loop is involved in the survival pathway of IL-3.     

骨桥蛋白在胰腺癌中的表达及其临床意义

目的:研究骨桥蛋白OPN在胰腺癌中的表达水平及其与临床病理特征的关系。方法:采用RT-PCR和免疫组织化学方法分别检测50例胰腺癌手术切除标本和20例癌旁正常胰腺组织中OPNmRNA及其蛋白水平的表达。结果:胰腺癌组织中OPNmRNA高表达率为90%(45/50)明显高于其在癌旁正常胰腺组织中的表达15%(3/20),差异有统计学意义(P<0.01);OPN蛋白的阳性率为86.0%(43/50),明显高于癌旁正常胰腺组织中的表达30%(6/20),差异有统计学意义(P<0.01);OPN在胰腺癌组织中表达与其淋巴结转移明显相关(P<0.05)。结论:OPN在胰腺癌中的过表达对胰腺癌的生长、浸润及转移起重要作用。     

Cell type-specific post-translational modifications of mouse osteopontin are associated with different adhesive properties

Osteopontin (OPN) is a highly modified integrin-binding protein found in all body fluids. Expression of OPN is strongly correlated with poor prognosis in many different human cancers, suggesting an important but poorly understood role for this protein in tumorigenesis and metastasis. The protein exists in a number of different isoforms differing in the degree of post-translational modifications that are likely to exhibit different functional properties. This study examines for the first time the post-translational modifications of OPN from transformed cells and the effects of these modifications on cell biology. We have characterized the complete phosphorylation and glycosylation patterns of OPN expressed by murine ras-transformed fibroblasts (FbOPN) and differentiating osteoblasts (ObOPN) by a combination of mass spectrometric analyses and Edman degradation. Mass spectrometric analysis showed masses of 34.9 and 35.9 kDa for FbOPN and ObOPN, respectively. Enzymatic dephosphorylation, sequence, and mass analyses demonstrated that FbOPN contains approximately four phosphate groups distributed over 16 potential phosphorylation sites, whereas ObOPN contains approximately 21 phosphate groups distributed over 27 sites. Five residues are O-glycosylated in both isoforms. These residues are fully modified in FbOPN, whereas one site is partially glycosylated in ObOPN. Although both forms of OPN mediated robust integrin-mediated adhesion of mouse ras-transformed fibroblasts, the less phosphorylated FbOPN mediated binding of MDA-MD-435 human tumor cells almost 6-fold more than the heavy phosphorylated ObOPN. These results strongly support the hypothesis that the degree of phosphorylation of OPN produced by different cell types can regulate its function.