Study of HSP70, PDI and GnRH receptor in Scylla paramamosain

拟穴青蟹Scyllaparamamosain（Estampador,1949）是我国东南沿海4种青蟹中的主要优势种，重要的海洋经济蟹类之一。本研究采用分子生物学和免疫学技术对拟穴青蟹的一些功能蛋白——热休克蛋白70（heatshockprotein70，HSP70）、蛋白质二硫键异构酶（proteindisulfideisomerase，PDI）和促性腺激素释放激素受体（gonadotropin-releasinghormonereceptor，GnRH-R）进行研究，旨在证明这些蛋白在拟穴青蟹体内的存在，了解其分子结构和分布，为拟穴青蟹生殖调控机制的研究以及水产养殖的发展提供基础资料。所涉及...Scylla paramamosain (Estampador, 1949), an important economic marine crab, is the dominant species in the four species of genus Scylla in the coast of southeasten China. The existence, molecular structure and tissue expressing of three different kinds of function protein, including heat shock protein 70 (HSP70), protein disulfide isomerase (PDI) and gonadotropin-releasing hormone receptor (GnRH-R)...学位：理学硕士院系专业：海洋与环境学院海洋学系_海洋生物学学号：2112005130217

Study of HSP70, PDI and GnRH receptor in Scylla paramamosain

拟穴青蟹Scyllaparamamosain（Estampador,1949）是我国东南沿海4种青蟹中的主要优势种，重要的海洋经济蟹类之一。本研究采用分子生物学和免疫学技术对拟穴青蟹的一些功能蛋白——热休克蛋白70（heatshockprotein70，HSP70）、蛋白质二硫键异构酶（proteindisulfideisomerase，PDI）和促性腺激素释放激素受体（gonadotropin-releasinghormonereceptor，GnRH-R）进行研究，旨在证明这些蛋白在拟穴青蟹体内的存在，了解其分子结构和分布，为拟穴青蟹生殖调控机制的研究以及水产养殖的发展提供基础资料。所涉及...Scylla paramamosain (Estampador, 1949), an important economic marine crab, is the dominant species in the four species of genus Scylla in the coast of southeasten China. The existence, molecular structure and tissue expressing of three different kinds of function protein, including heat shock protein 70 (HSP70), protein disulfide isomerase (PDI) and gonadotropin-releasing hormone receptor (GnRH-R)...学位：理学硕士院系专业：海洋与环境学院海洋学系_海洋生物学学号：2112005130217

Immunological Recognition of Gonadotropin-releasing Hormone Receptor(GnRH-R) in the Mud Crab,Scylla paramamosain

促性腺激素释放激素(gnrH)及其受体(gnrH-r)对生殖具有重要的调控作用.迄今无脊椎动物gnrH及gnrH-r的研究尚少.本研究采用兔抗人gnrH-r、兔抗果蝇gnrH-r和兔抗海鞘gnrH-r的抗体,应用免疫印迹和免疫共沉淀技术对拟穴青蟹(SCyllAPArAMAMOSAIn)gnrH-r进行了免疫识别,所得结果如下:1)经免疫印迹检测,拟穴青蟹脑、胸神经团、视神经节和精巢中共有的免疫阳性条带分子质量为45--55 ku.脑和胸神经团中另有一条36 ku条带;在精巢中,兔抗人gnrH-r和兔抗果蝇gnrH-r抗体只有一条28 ku条带,而兔抗海鞘gnrH-r抗体显示有28,36和30 ku的条带.2)利用免疫共沉淀技术,分离出与gnrH-r抗体相结合的两种物质,分子质量分别为38.1和54.0 ku,分别与哺乳动物和非哺乳动物gnrH-r的分子质量接近.该发现提示拟穴青蟹体内存在gnrH-r,为深入了解甲壳动物生殖调控机理提供了理论依据.Gonadotropin-releasing hormone(GnRH) and its receptor(GnRH-R)play an important role in the regulation of reproduction.To date,there has been little study on the GnRH and GnRH-R in invertebrates.In this study,with three GnRH-Rs of rabbit antibody against different species,including human,fruit fly and sea squirt,Western blotting and co-immunoprecipitation were applied to test the GnRH-R in mud crab,Scylla paramamosain.The common immunoreactive bands,with the molecular mass between 45 and 55 ku,appeared in the brain,thoracic ganglion,eyetalk and testes.Another band with 36 ku was found in the brain and thoracic ganglion.And in the testes,there was only a band with 28 ku using the antibody against human and fruit fly,while using rabbit antibody against sea squirt,there were bands with 28,36 and 30 ku.Two substance combined to GnRH-R were separated by co-immunoprecipitation,with the molecular mass of 38.1 and 54.0 ku,which were close to the mammal and non-mammal,respectively.This study shows that GnRH-R occurs in mud crab,which is helpful to further understanding of the regulation of reproduction in crustaceans.国家自然科学基金项目(41076081

Molecular Cloning and Tissue Expression of HSP70 from the Mud Crab,Scylla paramamosain

热休克蛋白70(HSP70)是一种重要的功能蛋白.利用rACE和基因克隆等分子生物学技术,获得拟穴青蟹(SCyllA PArAMAMOSAIn)HSP70全长CdnA序列,全长共2 189 bP,包括110 bP的5′uTr(unTrAnSlATEd rEgIOnS)、126 bP的3′uTr和一个1 953 bP的开放阅读框(Orf).Orf可编码650个氨基酸残基,总分子质量约为71.2 ku,PI为5.38.同源蛋白的比较结果显示,拟穴青蟹HSP70序列与其他一些物种具有很高相似性,推测HSP70基因具有很高的遗传保守性,而基于HSP70氨基酸序列比对而绘制的系统树基本能够反映出各物种间的进化关系.以rT-PCr法检测雌性拟穴青蟹一些组织中的HSP70表达,结果显示各待测组织中均能扩增得到HSP70,说明HSP70在拟穴青蟹为组成型表达.其中,HSP70在卵巢中表达量最高,其次为脑神经节,推测这与HSP70作为分子伴侣的功能相关.Heat shock protein 70(HSP 70) is an in evolutionarily highly conserved protein,with a variety of biological functions.A full-length cDNA of HSP70 was cloned from Scylla paramamosain using RACE and gene cloning method in this study.The full-length HSP70 cDNA was 2 189 bp,including a 5′untranslated region(UTR) of 110 bp,a 3′UTR of 126 bp,and an open reading frame(ORF) of 1 953 bp nucleotides.The ORF encoded a polypeptide of 650 amino acids with calculated molecular mass of 71.2 ku and theoretical point of 5.38.Amino acid sequence alignment showed the S.paramamosain HSP70 had high homology with that in some model animals.It suggested HSP70 was highly conservative.Phylogenetic tree based on comparison of HSP70 proteins,was consistent with the evolution relationship.The tissue expressions in 8 different tissues were tested by RT-PCR.The results showed that the expected PCR products were amplified from every tested tissues.It suggested that HSP70 can be constitutively expressed in S.paramamosain.The highest expression of HSP70 was found in ovary,the following was in brain,this was possibly related with the functions of HSP70.国家自然科学基金项目(4040603041076081

企业创新网络中关系强度、吸收能力与创新绩效的关系研究

有关研究表明,在创新网络中,企业创新绩效受到外部网络关系和自身吸收能力两方面的影响,但网络关系的强弱对企业创新绩效的影响在学术界尚未达成共识。在中国关系社会的背景之下,本文构建了网络关系强度、企业吸收能力和创新绩效三者之间的关系理论模型,并以中国278家制造业企业为样本进行了实证研究。研究结果表明,创新网络关系强度和企业吸收能力均对企业创新绩效存在显著的正向影响,且企业吸收能力在网络关系强度和企业创新绩效之间起不完全中介作用。同时,本文检验了企业性质、产业类型和地域分布的影响,发现在私营企业、传统制造业企业和位于华东、华北地区的企业中,创新网络关系强度对创新绩效没有直接影响,但可以通过吸收能力对创新绩效产生间接影响

Humic acid from leonardite for cadmium adsorption and potential applications

以碱溶酸析＂法从新疆风化煤中提取的胡敏酸为研究对象,对其理化性质和表面形态进行表征,并通过吸附试验探究反应时间、溶液pH、镉离子（Cd（2＋））质量浓度对胡敏酸吸附Cd（2＋）的影响.结果表明：风化煤提取的胡敏酸碳元素质量分数高达58.68%,羧基的质量摩尔浓度为5.81mol/kg,等电点为2.7;该胡敏酸含Cd量为0.15mg/kg,符合国家土壤环境质量标准.胡敏酸对Cd（2＋）的吸附在8h内达到平衡,吸附量随Cd（2＋）质量浓度（0～100mg/L）和溶液pH升高而增加,到pH=6.0时最大,之后胡敏酸开始溶解导致吸附量降低.Langmuir方程比Freundlich方程能更好地拟合胡敏酸对Cd（2＋）的吸附等温线,显示出单分子层吸附的特点.在pH=5.0时,胡敏酸对Cd（2＋）的饱和吸附量达137.37mg/g,相当于用去了酸度系数（pKa）为3的羧基含量的71%.在pH=4.3、Cd（2＋）初始质量浓度为80mg/L的同等条件下,新疆风化煤提取的胡敏酸对Cd（2＋）的吸附量为86.97mg/g,高于国际腐殖质协会胡敏酸标样1R106H对Cd（2＋）的吸附量（73.49mg/g）.风化煤来源广、储量大、价格低,以它为原料制备获得的胡敏酸产量高、吸附能力强、环境友好、施用安全,有望作为吸附剂用于含重金属废水处理,以及作为钝化剂和土壤调理剂用于重金属污染土壤的修复

Cloning and Sequence Analysis of Scylla paramamosain Protein-disulfide Isomerase Conserved Domain

【中文摘要】 蛋白二硫键异构酶(PDI)是真核生物重要的多功能蛋白,其基因结构在虾蟹类尚未报道.从拟穴青蟹Scylla pa-ramamosain(Estampador,1949)脑组织中分离纯化总RNA,经逆转录得到cDNA第一条链,并以之为模板利用RACE(rapid-amplification of cDNA ends)技术,扩增出一条950 bp的cDNA.将所得cDNA克隆到质粒载体pTZ57R/T,转化大肠杆菌DH5α细胞并对筛选的阳性克隆测序.通过与目前数据库中序列的比较,在此cDNA编码蛋白中发现其中101个氨基酸与其他物种已知蛋白二硫键异构酶(PDI)中的PDI_a_PDI_a′_C保守区域相似率在64%~79%之间.确定此cDNA编码拟穴青蟹PDI_a_PDI_a′_C保守区域. 【英文摘要】 The protein disulfide isomerase severs to be a multifunctional protein of great importance to the eukaryotes.The total RNAs isolated from the brain of Scylla paramamosainn(Estampador,1949) were reverse transcribed to the first strands of cDNA.A cDNA fragment of 950 bp was amplified from the first strands of cDNA by RACE(rapid-amplification of cDNA ends) method.This fragment was inserted into plasmid pTZ57R/T for screening and DNA sequencing.Compared with current sequences in the database,a 101-aa domain,whi...国家自然科学基金(40406030,40776084);福建省高等学校新世纪优秀人才支持计划资

一种用于重金属钝化的土壤调理剂及其制备方法

本发明涉及一种用于重金属钝化的土壤调理剂及其制备方法。该制备方法为：由富含腐殖酸的物料和强碱溶液混合，加热后提取的腐殖酸经联合絮凝方法得到用于重金属钝化的土壤调理剂。本发明利用分布广泛、储量丰富、廉价易得的褐煤和风化煤为原料，经环境友好过程提取，用首创的联合絮凝法制得用于重金属钝化的土壤调理剂。本发明的土壤调理剂由腐殖酸钙组成，富含羧基、羟基等功能基团，能有效吸附重金属，从而降低它们的生物有效性，可用于包括酸性土壤在内的重金属污染农田的调理和修复

Humic acid from leonardite for cadmium adsorption and potential applications

以碱溶酸析"法从新疆风化煤中提取的胡敏酸为研究对象,对其理化性质和表面形态进行表征,并通过吸附试验探究反应时间、溶液pH、镉离子(Cd~(2+))质量浓度对胡敏酸吸附Cd~(2+)的影响.结果表明:风化煤提取的胡敏酸碳元素质量分数高达58.68%,羧基的质量摩尔浓度为5.81mol/kg,等电点为2.7;该胡敏酸含Cd量为0.15mg/kg,符合国家土壤环境质量标准.胡敏酸对Cd~(2+)的吸附在8h内达到平衡,吸附量随Cd~(2+)质量浓度(0~100mg/L)和溶液pH升高而增加,到pH=6.0时最大,之后胡敏酸开始溶解导致吸附量降低.Langmuir方程比Freundlich方程能更好..