小菊锌指蛋白基因cDNA全长克隆及表达分析

目的：克隆菊花耐盐碱相关锌指蛋白基因,并进行盐胁迫和品种间差异的表达分析。方法：从大量菊花资源中筛选出抗盐碱品系小菊‘阳光’,利用RT—PCR从经150mmol／L碳酸钠处理的小菊‘阳光’叶片中分离得到一个锌指蛋白cDNA全长克隆,用Northern杂交检测其在不同盐处理和不同品种小菊中的表达。结果：获得了全长794bp的基因CmSTZF（GenBank接受号为DQ864730）,编码区为170个氨基酸残基。Blast分析表明,CmSTZF含有AN1型锌指结构,序列模式为C-X2-C—X（9—12）-C-X（1-2）-C-X4-C-X2-H-X5-H-X-C,由Cys^110-Cys^113-Cys^131-His^134及Cys^124-Cys^126-Cys^142-His^140分别围绕锌离子与其他氨基酸共同组成2个锌指四面体结构;在第67～76及第94～104氨基酸残基序列间存在核定位信号。同源性比较发现,CmSTZF与水稻OsISAPI具有54％的同源性,而二者的锌指保守区相似性达100％。Cluster分析表明,小菊CmSTZF锌指蛋白与水稻的2种逆境反应蛋白亲缘关系最近,归属同一类逆境功能蛋白。在150mmol／L碳酸钠胁迫下,耐盐小菊‘阳光’锌指蛋白表达量明显高于非耐盐小菊‘神韵’,表明CmSTZF锌指蛋白基因在盐碱胁迫下起重要的调控作用。结论：克隆了小菊耐盐碱相关的锌指蛋白基因CmSTZF,其在耐盐小菊‘阳光’中的表达量高于非耐盐小菊‘神韵’,这为小菊锌指蛋白基因CmSTZF耐盐碱功能分析奠定了基础。     

西伯利亚PsPIP1基因的克隆及其在NaHCO3胁迫下的表达

应用cDNA末端快速扩增（RACE）技术从西伯利亚蓼叶cDNA文库中克隆了质膜内在蛋白基因（PsPIP1的完整编码区cDNA序列（GenBank accession No．EU626398）,长度为1004bp,编码285个氨基酸。基于和其他植物水通道蛋白的氨基酸序列、推测的三维结构的比较以及系统进化分析结果,初步确定此基因为水通道蛋白基因家族中的PIP1亚族成员。RT-PCR结果显示,PsPIP1在西伯利亚蓼的地下茎、茎、叶中均有表达,叶中表达量最高,地下茎次之,茎中最低。在NaHCO3胁迫与去胁迫的过程中,此基因在地下茎、茎、叶中的表达模式也有较明显的差异。     

西伯利亚蓼PsPIP1基因的克隆及其在NaHCO3胁迫下的表达

应用cDNA末端快速扩增(RACE)技术从西伯利亚蓼叶cDNA文库中克隆了质膜内在蛋白基因(PsPIP1)的完整编码区cDNA序列(GenBank accession No. EU626398), 长度为1 004 bp, 编码285个氨基酸。基于和其他植物水通道蛋白的氨基酸序列、推测的三维结构的比较以及系统进化分析结果, 初步确定此基因为水通道蛋白基因家族中的PIP1亚族成员。RT-PCR结果显示, PsPIP1在西伯利亚蓼的地下茎、茎、叶中均有表达, 叶中表达量最高, 地下茎次之, 茎中最低。在NaHCO₃胁迫与去胁迫的过程中, 此基因在地下茎、茎、叶中的表达模式也有较明显的差异。     

耐盐杂草稻3个锌指蛋白基因家族的实时定量分析

利用在300余份来源于辽宁、吉林、黑龙江、内蒙古、江苏等地的杂草稻材料中筛选出耐高盐杂草稻材料WR03-12。通过RT-PCR的方法得到盐胁迫下WR03-12与盐敏感栽培稻‘越光’幼苗的cDNA第一链,对3个锌指蛋白基因家族的6个基因表达情况进行了荧光实时定量分析。结果表明,2个C2C2型锌指蛋白基因SRZ1与SRZ2受到高盐胁迫的负向诱导,WR03-12受负向诱导程度要小于‘越光’;2个TFIIIA型锌指蛋白基因ZFP18与ZFP245受到盐胁迫的正向诱导,WR03-12受诱导程度也小于‘越光’;具有A20锌指结构的基因AACZ1基因在越光中不受盐诱导,而在WR03-12中受短时间诱导后,第7天已经恢复到胁迫前水平。具有AN1锌指结构的基因AACZ2在‘越光’与WR03-12中均不受盐胁迫诱导,且表达水平没有显著差别。杂草稻WR03-12与‘越光’对于盐胁迫的应答机制可能在转录调控方面存在差别。     

小黑杨环锌指蛋白基因的克隆与表达分析

用cDNA-AFLP技术从小黑杨中克隆与盐胁迫反应相关的cDNA片段,进一步应用RACE技术克隆出具有完整开放读码框的小黑杨环锌指蛋白基因（PsnRZF）,该基因全长1061bp,其中5非翻译区为184bp,3非翻译区为82bp,开放读码框为795bp,编码264个氨基酸,预测蛋白的分子量为30.25kDa,理论等电点为8.04。实时定量PCR检测的结果显示,正常生长条件下该基因在根、茎、叶中都表达;NaCl胁迫下,该基因在根、茎、叶中的表达量升高。在叶中的表达量随着处理时间的延长而逐渐升高,胁迫处理后第6天表达量达到最高。     

逆境诱导水稻谷胱甘肽合成酶基因的表达研究

用RACE方法获得了全长的水稻谷胱甘肽合成酶(GS)基因的cDNA,并命名为OsGS(GeneBankaccession No.:AY453405)。该cDNA全长1 892 bp,编码一个由540个氨基酸组成的多肽,预测其氨基端(N端)含有一段定位叶绿体的信号肽。比较水稻基因组定位结果表明OsGS基因位于水稻12号染色体短臂上,转录区全长6 321 bp,由12个外显子和11个内含子组成。通过RT-PCR对OsGS在水稻正常生长条件和逆境条件下的表达进行了研究。结果表明,在正常生长条件下,OsGS在水稻幼苗的根和叶以及抽穗期水稻的根中表达;但不在抽穗期水稻的叶、茎和幼穗中表达,这显示OsGS在水稻中的表达具有发育和组织特异性。利用抽穗期水稻的叶片为材料,经高温、干旱和重金属逆境处理后,OsGS在抽穗期叶片中的转录被诱导表达;而在盐、低温、伤害逆境下则不被诱导。在轻度和中度干旱胁迫4 h后OsGS基因可被诱导表达。外源ABA处理也能够提高OsGS的转录水平,这显示OsGS可能是依赖ABA信号途径的环境胁迫诱导基因。     

小黑杨转录因子PsnbHLH162基因在盐和低温胁迫下应答分析

为揭示小黑杨(Populus simonii×P. nigra)在面对非生物胁迫时,转录因子PsnbHLH162在植物体内发挥的作用,同时探究该基因在植物体内的信号转导过程,进而为未来获取优良的抗逆树种提供理论基础。以小黑杨为原材料,克隆获取PsnbHLH162基因,对目的基因和启动子进行生物信息学分析;之后用150 mmol·L^-1 NaCl、4℃低温分别对野生型小黑杨进行胁迫处理,利用荧光定量PCR,分析基因响应非生物胁迫的功能。结果显示：PsnbHLH162 cDNA基因片段长537 bp,基因N端内含1个高度保守的HLH结构域。该基因表达蛋白是不含跨膜区域的稳定的亲水性蛋白,其定位在细胞核内且没有转录激活活性。启动子区域内含多种ABA应答、生长素应答、光应答和circadian元件,证实此基因参加非生物胁迫应答。荧光定量PCR结果表明在盐胁迫下,与茎、叶组织相比,基因在根组织的表达量最高;在低温胁迫下,与叶、根组织相比,基因在茎组织的表达量最高。发现野生植株内,PsnbHLH162能被盐、低温诱导表达。     

西番莲PeERG基因克隆及其表达模式分析

ERA(Eecherichia coli Ras-like protein)蛋白是与已知异三聚体G蛋白和小分子G蛋白不同的一种新的GTP结合蛋白。为了在木本植物中开展其同源基因ERG(ERA-like GTPase)克隆和功能验证的相关研究,该文首次在西番莲新品种‘平塘1号’中采用cDNA末端快速克隆(RACE)技术克隆鉴定1个ERG基因。结果表明:西番莲PeERG基因cDNA全长为1 518 bp,包括1 260 bp的开放阅读框、38 bp的5'-端非翻译区和220 bp的3'-端非翻译区,该基因编码蛋白由420个氨基酸残基组成,其二级结构含有丰富的α-螺旋和延伸链。PeERG蛋白不含跨膜区域,也不存在信号肽酶切位点,既在其N端有典型的GTPase保守结构域(GTPase domain)又在其C端有独特的RNA结合结构域(KH domain)。系统进化树分析表明,西番莲PeERG蛋白和水稻OsERG1、拟南芥AtERG1、大肠杆菌ERA位于同一进化分枝。实时定量PCR检测揭示PeERG基因在西番莲根、茎、叶、花、果中均有表达,叶中表达最高;同时该基因响应低温胁迫信号,其表达呈动态变化模式。该研究首次鉴定和描述了木本植物西番莲的ERG基因,为深入挖掘西番莲特异基因资源提供参考,也有助于进一步探究ERG基因在植物中的生物学功能及其作用机制。     

番茄两个盐胁迫响应基因的cDNA克隆及其表达分析

根据前期耐盐芯片研究提供的两条EST序列设计引物,利用RACE技术从番茄耐盐品种Edkawi中克隆了其5’和3’片段,并拼接成全长cDNA,分别命名为SISRGl和SISRG2。两个基因序列在GenBank中的登录号为EU670751和EU670752,其大小分别为1300bp和1810bp,编码蛋白分别为309和499个氨基酸。半定量RT,PCR表明SISRGl在番茄茎、叶、花中表达较强,在所检测的其它组织中表达很弱,SISRG2在叶和花中表达量最高,其次为茎和根,在果实中表达微弱。盐胁迫表达谱结果显示SISRGl在盐处理的Edkawi中缓慢增强,SISRG2则在盐胁迫后表达迅速增强,在未进行盐胁迫的对照中,两个基因的表达趋势均为减弱。本研究为番茄抗逆研究提供了新的候选基因资源。     

茶树CsGIF1基因的克隆及其对非生物胁迫的响应分析

GRF-INTERACTING FACTOR(GIF)基因是植物叶发育相关的一类重要调控因子,调节植物叶器官的发育。该研究采用RT-PCR方法从茶树‘龙井43’的叶片cDNA中克隆得到CsGIF1基因,并利用荧光定量PCR分析了高温(38℃)、低温(4℃)、干旱(200g·L-1 PEG)、盐胁迫(200mmol·L-1 NaCl)下CsGIF1基因的表达水平,以明确CsGIF1基因对非生物胁迫的应答特性,为茶树CsGIF1基因的逆境调控以及功能研究奠定基础。结果表明:(1)CsGIF1基因长666bp,编码221个氨基酸,具有高度保守的SNH结构域;CsGIF1蛋白为亲水性蛋白,理论相对分子质量为23 380,理论等电点为6.30;酸性氨基酸、碱性氨基酸、芳香族氨基酸和脂肪族氨基酸所占比例分别为7%、9%、5%和13%;蛋白质二级结构显示,茶树CsGIF1蛋白由38.01%的α-螺旋、10.41%的β-折叠、12.22%的延伸主链和39.37%的随机卷曲组成。(2)qRT-PCR分析显示,茶树CsGIF1基因对于4种非生物胁迫均有响应,但不同处理响应不同。其中在胁迫2h时,高温和低温胁迫下,CsGIF1基因相对表达量显著增加,分别为对照的3.54和5.69倍,且高低温胁迫下,CsGIF1基因响应明显大于干旱和盐胁迫。在高温、低温、干旱、盐等不同胁迫处理下,茶树‘龙井43’中CsGIF1基因的表达水平与对照相比均差异明显。     

Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.     

Molecular cloning and characterization of a novel human gene (NESCA) which encodes a putative adapter protein containing SH3

A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1990 bp and has a predicted open reading frame encoding 433 amino acids. It possesses an Src homology 3 (SH3) motif, a leucine zipper motif and no catalytic domain, suggesting that it seems to be an adapter protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 1q21-22.     

CD44 interaction with tiam1 promotes Rac1 signaling and hyaluronic acid-mediated breast tumor cell migration

In this study we have explored the interaction between CD44 (the hyaluronic acid (HA)-binding receptor) and Tiam1 (a guanine nucleotide exchange factor) in metastatic breast tumor cells (SP1 cell line). Immunoprecipitation and immunoblot analyses indicate that both the CD44v3 isoform and the Tiam1 protein are expressed in SP1 cells and that these two proteins are physically associated as a complex in vivo. Using an Escherichia coli-derived calmodulin-binding peptide-tagged Tiam1 fragment (i.e. the NH(2)-terminal pleckstrin homology (PHn) domain and an adjacent protein interaction domain designated as PHn-CC-Ex, amino acids 393-738 of Tiam1) and an in vitro binding assay, we have detected a specific binding interaction between the Tiam1 PHn-CC-Ex domain and CD44. Scatchard plot analysis indicates that there is a single high affinity CD44 binding site in the PHn-CC-Ex domain of Tiam1 with an apparent dissociation constant (K(d)) of 0.2 nM, which is comparable with CD44 binding (K(d) = approximately 0.13 nM) to intact Tiam1. These findings suggest that the PHn-CC-Ex domain is the primary Tiam1-binding region for CD44. Most importantly, the binding of HA to CD44v3 of SP1 cells stimulates Tiam1-catalyzed Rac1 signaling and cytoskeleton-mediated tumor cell migration. Transfection of SP1 cells with Tiam1cDNA promotes Tiam1 association with CD44v3 and up-regulates Rac1 signaling as well as HA/CD44v3-mediated breast tumor cell migration. Co-transfection of SP1 cells with PHn-CC-Ex cDNA and Tiam1 cDNA effectively inhibits Tiam1 association with CD44 and efficiently blocks tumor behaviors. Taken together, we believe that the linkage between CD44v3 isoform and the PHn-CC-EX domain of Tiam1 is required for HA stimulated Rac1 signaling and cytoskeleton-mediated tumor cell migration during breast cancer progression.     

Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211bp, consisting of a 5'-terminal untranslated region (UTR) of 72bp, a 3'-terminal UTR of 77bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16h after injury and injection of bacteria.     

Use of the polymerase chain reaction to clone and sequence a cDNA encoding the bovine alpha 3 chain of type IV collagen

A novel type IV collagen, alpha 3(IV), has previously been isolated from a collagenase digest of bovine and human glomerular and lens basement membranes. The cloning and sequencing of a cDNA encoding the alpha 3(IV) chain is described here. Using the polymerase chain reaction, with primers derived from the known 27-residue bovine alpha 3(IV) amino acid sequence, a 68-base pair bovine genomic fragment (KEM68) which encodes the known peptide sequence, was synthesized. KEM68 was then used to screen a bovine lens cDNA library and a 1.5-kilobase partial cDNA clone obtained, encoding 471 residues of the bovine alpha 3(IV) chain: 238 residues from the triple helical collagenous domain and all 233 residues of the noncollagenous domain. The collagenous repeat sequence has three interruptions, coinciding with those in the alpha 1(IV) chain. The noncollagenous domain has 12 cysteine residues in identical positions to those of other type IV collagens and 71, 61, and 70% overall similarity with the human alpha 1(IV), alpha 2(IV), and alpha 5(IV) chains. The noncollagenous domain of alpha 3(IV) is of particular interest as it appears to be the component of glomerular basement membrane that reacts maximally with the Goodpasture antibody. Furthermore, such antigenicity is absent from collagenase digests of the glomerular basement membrane of some patients with Alport syndrome. The alpha 3(IV) cDNA clone described here now permits study of the molecular pathology of COL4A3 in Alport syndrome.     

Primary structure of nonspecific crossreacting antigen (NCA), a member of carcinoembryonic antigen (CEA) gene family, deduced from cDNA sequence

A cDNA containing the entire coding region for a member of carcinoembryonic antigen (CEA) gene family has been cloned from cDNA library of HLC-1 cells by immunochemical screening with the antibody specific to nonspecific crossreacting antigen (NCA). The cDNA encodes a precursor form of a polypeptide consisting of a 34-residue signal sequence, a 108-residue N-terminal (N-) domain, a 178-residue domain (NCA-I domain) and a 24-residue domain rich in hydrophobic amino acids (M-domain). Each domain has a distinct but homologous amino acid sequence to that of the corresponding domain of CEA. Unlike the coding sequences, the 3'-untranslated sequences differ markedly in the NCA and CEA cDNAs facilitating the preparation of probes that will discriminate between nucleotide sequences for CEA and NCA.     

3BP-1, an SH3 domain binding protein, has GAP activity for Rac and inhibits growth factor-induced membrane ruffling in fibroblasts.

The SH3 binding protein, 3BP-1, was originally cloned as a partial cDNA from an expression library using the Abl SH3 domain as a probe. In addition to an SH3 binding domain, 3BP-1 displayed homology to a class of GTPase activating proteins (GAPs) active against Rac and Rho proteins. We report here a full length cDNA of 3BP-1 which extends the homology to GAP proteins previously noted. 3BP-1 functions in vitro as a GAP with a specificity for Rac-related G proteins. Microinjection of the 3BP-1 protein into serum-starved fibroblasts produces an inhibition of platelet-derived growth factor (PDGF)-induced membrane ruffling mediated by Rac. Co-injection of 3BP-1 with an activated Rac mutant that is unresponsive to GAPs, counter-acts this inhibition. 3BP-1 does not show in vitro activity towards Rho and, in agreement with this finding, microinjection of 3BP-1 into fibroblasts has no effect on lysophosphatidic acid (LPA)-induced stress fiber assembly mediated by Rho. Thus 3BP-1 is a new and specific Rac GAP that can act in cells to counter Rac-mediated membrane ruffling. How its SH3 binding site interacts with its GAP activity remains to be understood.     

Purification, cDNA cloning and characterization of the vascular apoptosis-inducing protein, HV1, from Trimeresurus flavoviridis.

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification and cDNA cloning from Crotalus atrox of a vascular apoptosis-inducing protein (VAP1) that specifically induces apoptosis in vascular endothelial cells. We report here the purification and cDNA cloning of another vascular apoptosis-inducing protein, HV1, from crude venom of Trimeresurus flavoviridis. The protein, namely HV1, was purified as an inducer of apoptosis in cultured vascular endothelial cells. HV1 was a homodimeric protein with a molecular mass of 110 kDa. HV1 cDNA encoded a protein with 612 amino-acid residues. The amino-acid sequence predicted from the cDNA was highly homologous to VAP1. The amino-acid sequence of HV1 indicated that HV1 belongs to the metalloprotease/disintegrin family, and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD, replaces the RGD sequence that has frequently been found in such domains. This replacement also occurs in VAP1. Our results indicate HV1 as the first identified homolog of VAP1.