海洋球石藻(Emiliania huxleyi)病毒硫氧还蛋白(TRX)基因的克隆及生物信息学分析

首次从海洋球石藻病毒(Emiliania huxleyi virus-EhV99B1)中克隆了硫氧还蛋白(Trx)基因(该序列已提交GenBank,登录号:GU109280)。系统进化关系分析表明:EhV99B1-Trx与GenBank中已报道的EhV86-Trx(NC_007346)有很高的同源性,核酸及其推导的氨基酸序列的同源性分别为98%和100%,而与其它物种的Trx序列同源性仅为9.8%-18.8%。采用生物信息学方法和工具分析了EhV99B1-Trx的生化参数,预测了该蛋白的高级结构。结果表明:(1)EhV99B1-Trx基因的开放阅读框全长591bp,编码196个氨基酸,蛋白的相对分子量为22.1kDa,理论等电点为5.27;(2)EhV-99B1-Trx N端蛋白结构域具有保守的Cys-Gly-Pro-Cys(CGPC)硫氧还蛋白氧化还原活性位点氨基酸基序;(3)对该蛋白二、三级结构分析预测结果进一步证实EhV99B1-Trx基因编码的为一种新型的硫氧还蛋白家族成员。     

青蛤(Cyclina sinensis)金属硫蛋白及硫氧还蛋白基因的克隆与表达分析

利用构建的cDNA文库及高通量测序方法,获得金属硫蛋白及硫氧还蛋白基因全长。结果表明,青蛤金属硫蛋白和硫氧还蛋白的cDNA全长分别为776bp和804bp,金属硫蛋白基因编码74个氨基酸,包含7个CXC特征结构;硫氧还蛋白基因编码165氨基酸,包含5个氨基酸构成的活性中心。采用实时定量PCR方法分析了两种基因在Cd2+胁迫下的表达变化,结果显示,金属硫蛋白基因在Cd2+胁迫下6—12hmRNA表达量急剧上升,与对照组有显著性差异(P<0.01)。硫氧还蛋白基因在Cd2+胁迫下mRNA在6h明显升高,在6—24h与对照组出现显著性差异(P<0.05)。说明Cd2+能够诱导青蛤金属硫蛋白和硫氧还蛋白基因表达的时序性升高,二者的转录过程反映了贝类抵御重金属胁迫的分子调控过程。     

海湾扇贝(Argopecten irradians)金属硫蛋白基因的克隆与分析

金属硫蛋白是一种普遍存在于生物体内的低分子量、半胱氨酸含量丰富、易于被外界刺激诱导的金属结合蛋白。采用表达序列标签法,结合cDNA末端快速扩增技术,首次获得了海湾扇贝金属硫蛋白(AiMT)的全长cDNA序列。该序列全长787bp,5′UTR(UntranslatedRegion)为79bp,3′UTR为270bp,开放阅读框(OpenReadingFrame,ORF)长度为438bp,可编码145个氨基酸。在其编码的氨基酸序列中半胱氨酸含量丰富,甘氨酸含量也较高,芳香族氨基酸含量低,不含组氨酸,存在有无脊椎动物和软体动物金属硫蛋白的特征序列CKCXXX-CXCX,C-末端的氨基酸序列也符合软体动物金属硫蛋白标签序列C-x-C-x(3)-C-T-G-x(3)-C-x-C-x(3)-C-x-C-K。序列特征分析表明,该序列具备金属硫蛋白的典型特征,是金属硫蛋白家族的成员。     

瘤背石磺表皮生长因子基因的克隆、结构及进化分析

首次在瘤背石磺(Onchidium struma)中克隆得到一种新的表皮生长因子(EGF)的cDNA序列全长。EGF基因cDNA的全长为1158bp,命名为Os-egf1,其中开放阅读框长度为846 bp,编码一条包含281个氨基酸残基的多肽链。根据氨基酸序列比对和结构域分析结果发现其含有2个保守的EGF结构域和1个EGF-like结构域,每个结构域中均包含至少6个半胱氨酸残基,且形成CX7 CX4-5 CX10-13CXCX8 C结构,其结构域由C1~C3、C2~C4和C5~C6之间形成的3个二硫键维持,符合表皮生长因子家族及其相关蛋白的特征结构域,但其余氨基酸序列与现有相关基因差异较大,推测可能是一种新的EGF-like基因。利用MEGA6.0软件构建Os-egf1与EGF家族相关蛋白的系统进化树,表明EGF家族蛋白具有一定的物种特异性。     

蛋白核小球藻核酮糖-1,5-二磷酸羧化/加氧酶小亚基部分基因序列的克隆与分析

以单细胞绿藻蛋白核小球藻(Chlorella pyrenoidosa)(藻株编号为NMBluh015-1)为实验材料,利用几种绿藻中的核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)的小亚基(rbcS)的保守序列,设计简并引物,克隆到rbcS的一条cDNA(245 bp)和3条DNA序列(其编号为CP1、CP2和CP3,长度依次为1425 bp、810 bp、705 bp)。序列比较结果表明,该蛋白核小球藻rbcS cDNA核苷酸序列与普通小球藻(C.vulgaris)、杜氏藻(Dunaliella tertiolecta)(rbcS1和rbcS2)及蛋白核小球藻"太阳小球藻"株的相似性依次为93%、92%(91%)和90%,而其编码的氨基酸序列与杜氏藻相似性最高(80%);3条rbcS DNA序列与绿藻及高等植物rbcS部分序列表现了较高的同源性,其中2条DNA(CP2和CP3)部分区段同源性很高。实验结果表明扩增得到的cDNA和DNA序列为蛋白核小球藻rbcS基因。     

长牡蛎细胞周期调控关键基因cyclin B3 的克隆及其在性腺发育中的作用

首次在长牡蛎（Crassostrea gigas）中克隆得到细胞周期蛋白B3（cyclin B3）的cDNA 全长序列和基因组结构序列。cyclin B3 基因cDNA 全长2383 bp, 其中编码区长度为1293 bp, 编码一条含430 个氨基酸的多肽链, 氨基酸序列比对和结构域分析均表明其为其他物种cyclin...     

坛紫菜别藻蓝蛋白α,β亚基基因的克隆和序列分析

以野生坛紫菜色素体DNA为模板, 通过PCR扩增获得编码坛紫菜别藻蓝蛋白α亚基和β亚基的序列及两者之间的间隔序列,该序列全长为1 055 bp,其中编码α和β亚基的序列均为486 bp,间隔序列为83 bp.该序列与GenBank收录的其他4种红藻相关序列的同源性在75.6%~87.4%,与2种蓝藻的同源性分别为66.9%,68.5%,其中编码区同源性更高.     

硫氧还蛋白及其相关蛋白的研究进展

细胞的氧化还原状态随细胞内各种生理过程发生变化,例如,正在增殖的细胞与分化细胞相比,更需要处于还原状态;细胞内氧化还原环境的变化对于细胞周期或细胞凋亡都十分重要。细胞内的氧化还原状态主要由细胞内氧化还原型辅酶如NAD（P）H—NAD（P）和还原型谷胱甘肽-氧化型谷胱甘肽（GSHGSSG）浓度决定。另外,硫氧还蛋白及其相关蛋白也是维持细胞内氧化还原状态的二硫键还原酶。     

海洋球石藻病毒(Coccolithovirus)硫氧还蛋白(Trx)在毕赤酵母(Pichia pastoris)中的表达及其活性分析

从实验室保存的pBS-Trx重组质粒中克隆球石藻病毒EhV-Trx基因,构建毕赤酵母重组表达载体pPIC9K-EhV-Trx,将重组质粒电转化毕赤酵母GS115,诱导分泌表达并对重组蛋白进行二硫键还原酶活性分析。结果表明,EhV-Trx基因开放阅读框为591bp,编码197个氨基酸;在毕赤酵母GS115中成功诱导表达重组EhV-Trx,经SDS-PAGE分析目的蛋白分子量约为27.8kDa;重组EhV-Trx具有二硫键还原酶的活性,能有效打开胰岛素A、B两条链的二硫键,有望开发成一种新型的硫氧还蛋白脱敏制剂应用于食品安全领域。     

缢蛏生长因子受体结合蛋白2基因克隆、时空表达及SNP筛查

为了探索生长因子受体结合蛋白2（GRB2）在缢蛏生长发育中的作用,本实验基于缢蛏转录组文库,利用SMART RACE技术克隆缢蛏GRB2（Sc-GRB2）基因的cDNA全长序列,分析其在不同组织和发育时期的表达差异,并在外显子中进行了SNP位点筛选。结果表明,Sc-GRB2基因cDNA全长1 223 bp,开放阅读框711 bp,编码236个氨基酸;氨基酸序列比对发现,缢蛏与文蛤、泥蚶等双壳贝类同源性较高（64%、59%）,而与其他物种的同源性为45%~58%,表明GRB2基因比较保守;不同组织的荧光定量PCR （qRT-PCR）结果显示,Sc-GRB2基因在缢蛏7个组织中均有表达,其中足中的表达量极显著地高于其他6个组织（P<0.01）;在8个发育时期的表达差异分析发现,该基因在稚贝期表达量极显著地高于其他7个时期（P<0.01）。SNP位点筛选结果表明,在Sc-GRB2基因的外显子区域共发现11个SNP位点。     

Characteristics and phylogeny of light-harvesting complex gene encoded proteins from marine red alga Griffithsia japonica

1 Introduction T he light-harvesting com plex (LH C ) com posedofphotosynthesis pigm ents and associated proteins arelocated on the thylakoid m em brane of chloroplast inhigherplants and algae. T he capture oflightenergy isthe first step in photosynthesis and is largely accom -plished by the pigm entprotein com plexes. To m axi-m ize the capture of light energy under various envi-ronm entalconditions, the LH C s w ith differentchro-m ophore com positions have evolved in differentpho-tosynth…     

Cloning, expression and characterization of serine palmitoyltransferase (SPT)-like gene subunit (LCB2) from marine Emiliania huxleyi virus (Coccolithovirus)

The authors have isolated and characterized a novel serine palmitoyltransferase(SPT)-like gene in marine Emiliania huxleyi virus(EhV-99B1).The open-reading frame(ORF) of EhV99B1-SPT encoded a protein of 496 amino acids with a calculated molecular mass of 96 kDa and Ip 6.01.The results of sequence analysis showed that there was about 31%-45% identity in amino acid sequence with other organisms.The maximum likelihood phylogenetic tree suggested that the EhV99B1-SPT gene possibly horizontally transferred from the eukaryote.Hydrophobic profiles of deduced amino acid sequences suggested a hydrophobic,globular and membrane-associated protein with five transmembrane domains(TMDs) motifs.Several potential N-linked glycosylation sites were presented in SPT.These results suggested that EhV99B1-SPT was an integral endoplasmic reticulum membrane protein.Despite lower sequence identity,the secondary and three-dimensional structures predicted showed that the "pocket" structure element composed of 2α-helices and 4βsheets was the catalytic center of this enzyme,with a typical conserved "TFTKSFG" active site in the N-terminal region and was very close to those of prokaryotic organisms.However,the N-terminal domain of EhV99B1-SPT most closely resembled the LCB2 catalysis subunit and the C-terminal domain most closely resembled the LCB1 regulatory subunit of other organisms which together formed a spherical molecule.This "chimera" was highly similar to the prokaryotic homologous SPT.For a functional identification,the EhV99B1-LCB2 subunit gene was expressed in Escherichia coli,which resulted in significant accumulation of new sphingolipid in E.coli cells.     

Expressed sequence tag analysis and cloning of trehalose-6-phosphate synthase gene from marine alga Laminaria japonica (Phaeophyta)

A high quality cDNA library was constructed from the brown alga Laminaria japonica,with the titer of 1.2×10 5 pfu/ml.The average insert size of the cDNA library is about 1.6 kb.From the cDNA library,591 cDNA clones were randomly selected and sequenced.As a result,574 EST(expressed sequence tag) sequences were generated.All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198.The cDNA library was screened with a α-32 p labeled 453 bp T P S gene probe,which is a partial sequence yielded from Porphyra yezoensis.Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L.japonica TPS cDNA sequence.After PCR amplification,sequencing and assembling,the entire ORF(open reading frame) sequence of the T P S gene was obtained,which was named LjTPS.LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons.The LjTPS gene was successfully expressed in E.coli and rice.The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the T P S gene function and mechanism to stress tolerance.     

一种新的海洋微藻病毒丝氨酸蛋白酶基因的克隆表达及其活性分析

从海洋球石藻Emiliania huxleyi病毒EhV99B1的基因组中克隆了丝氨酸蛋白酶(Sp)基因(GenBank登录号:KC161207),对该基因的开放阅读框(ORF)进行系统的生物信息学分析,并在大肠杆菌中融合表达,通过亲和层析法获得了纯化的重组Sp。结果表明:EhV99B1-Sp基因的ORF为1 110bp,编码368个氨基酸,蛋白相对分子质量为39.5kDa;该基因片段与GenBank中EhV86-Sp的同源性很高,核苷酸及其对应的氨基酸序列同源性分别为95%和97%,而与其他物种Sp序列的同源性仅为28%~32%,说明其可能是丝氨酸蛋白酶家族中的一个新成员;预测的二级结构特征显示EhV99B1-Sp的蛋白结构域中具有典型的LTAGHC(组氨酸活性位点区域)和AICNGDSGGPLF(丝氨酸活性位点区域)两个丝氨酸蛋白酶催化活性位点的氨基酸基序,是一个两次跨膜蛋白;将该基因在大肠杆菌中进行低温诱导表达,得到分子量为60kDa的重组蛋白,经鲤鱼肌肉丝氨酸蛋白酶(MBSP)抗体检测证实为Sp,且重组蛋白在大肠杆菌细胞中具有明显的生物学活性。本研究结果为进一步探讨EhV99B1-Sp在病毒与宿主相互作用过程中的调节作用及其功能与应用奠定基础。     

Protective immunity of orange-spotted grouper(Epinephelus coioids) against a nervous necrosis virus isolated from China,and determination of the complete sequences of the virus

1 IntroductionNervous necrosis virus( NNV) is one of theworldwide pathogens in cultured species and breed-ing from the late 1980s (Bovo et al.,1999; Tan etal., 2001; Breuil et al., 2001; Munday et al.,2002; Lin et al., 2001). The virus is isometric andnon…     

委内瑞拉产地龙须菜藻红蛋白基因的克隆及其系统学研究

本文报道了红藻 Gracilaria lemaneiformis委内瑞拉株的藻红蛋白基因的部分序列 ,将所得序列与其它红藻 - Rhodella violacea,Polysiphonia boldii,Griffithsia monolis,Porphyra tenera,Porphyra yezoensis及青岛产龙须菜的相应序列对齐后 ,进行了系统学研究。结果显示 ,同一属的藻红蛋白 α和 β亚基之间的间隔序列 ,从长度到核苷酸序列均非常相似 ,而同一科不同属或同一目的科间的该序列有很大的不同 ;两不同产地龙须菜的 PE基因在 β亚基上的转换多于颠换 ,说明 β亚基比 α亚基保守 ;委内瑞拉来源龙须菜与青岛产地龙须菜可能不属于同一物种 ,应为同属不同种关系 ;由藻红蛋白基因所得的系统树包括 3个与建立在形态标准上的遗传位置一致的分支 ;藻红蛋白基因序列可用于种间及更高地位的分子系统研究。     

Isolation of CYP2L2 and two other cytochrome P450 sequences from a spiny lobster, Panulirus argus, hepatopancreas cDNA library

Cytochrome P450 monooxygenases constitute an enzyme superfamily, with at least 74 known families. Members of CYP families 1–4 are important in the phase 1 metabolism of lipophilic xenobiotics, such as those found in contaminated marine environments. Previous studies (James et al. Arch. Biochem. Biophys. (1996) 329, 31–38) showed that a major form of P450 in spiny lobster, Panulirus argus, hepatopancreas was CYP2L1, a new sub-family, and that there was evidence for other P450 forms in hepatopancreas. We now report the sequence of a second member of this subfamily, named CYP2L2, present in P. Argus hepatopancreas. The deduced amino acid sequences of CYP2L1 and CYP2L2 share 54.7% sequence identity and an additional 13.6% of the sequences show conservative substitutions. Analysis of the sequences of CYP2L1, CYP2L2 and other representative CYP2 family members (from rat and mouse sub-families 2A, 2B, 2D and 2E) showed that the crustacean sequences clustered together. In addition to CYP2L2, cDNA clones of 66 to 117 base pairs from the 5′ coding region of two more P450 isoforms were isolated from the spiny lobster cDNA library. The deduced amino acid sequence of one of these additional cDNA clones was identical to the first 22 amino acids of the N-terminal sequence of a P450 protein previously isolated from hepatopancreas microsomes. These studies confirm earlier biochemical evidence that the hepatopancreas contains multiple forms of cytochrome P450.     

合浦珠母贝iPS细胞相关的转录因子的克隆及分析

转录因子Sox2、Oct4、c-Myc和KLF4在iPS细胞研究中具有重要作用,将它们加入体外培养的细胞中,可以诱导体细胞去分化成为多能干细胞。作者通过RACE-PCR技术从合浦珠母贝(Pinctada fucata)的外套膜组织中克隆获得了3个转录因子——c-Myc、Sox2及KLF4的cDNA。c-Myc全长1585bp,开放阅读框的长度为1131bp,编码376个氨基酸,蛋白结构分析表明它具有保守的HLH和Myc-N结构域。Sox2全长1908bp,开放阅读框长度为990bp,编码329个氨基酸,蛋白结构分析它具有保守的HMG-box和Soxp结构域。KLF4全长2268bp,开放阅读框长624bp,编码207个氨基酸,预测该蛋白具有两个zf-H2C2_2结构域。BLAST分析它们均与太平洋牡蛎的相关蛋白具有较高同源性,说明其与太平洋牡蛎亲缘关系更近。RT-PCR实验发现这3个基因在合浦珠母贝外套膜、足、生殖腺、内脏团、闭壳肌和鳃6种组织中均有表达,表明它们是非常保守的转录因子。本研究对于合浦珠母贝细胞系建立和研究具有启发意义。