Identification and Analysis of Differentially Expressed Genes in Haemocytes of Variously Colored Abalone (Haliotis diversicolor Reeve,1846) Challenged with Bacteria

鲍以其丰富的营养价值在民间一直被认为是与鱼翅、燕窝齐名的营养食品。养殖鲍市场价格高且稳定，销路畅通，利润空间大，深受水产养殖户的欢迎，是我国重要的海水养殖经济物种。杂色鲍(HaliotisdiversicolorReeve,1846)隶属于软体动物门，腹足纲，前鳃亚纲，原始腹足目，鲍科，是我国南方鲍种,因其生长迅速，一直在南方沿海海域得到广泛的养殖。但近年来由于养殖海域水质影响及养殖鲍种自身退化，使得人工养殖杂色鲍抗病力低，时有疫病发生，给养鲍业带来很大的经济损失。因此，寻求免疫防治是保障鲍养殖业健康发展的重要措施，但迄今有关鲍免疫机制的研究报道较少。本项研究应用抑制性差减杂交(SSH)技术筛...Abalones are one of the largest marine gastropod mollusks and they are a economically important seafood in aquaculture worldwide. However, bacterial epidemic infection has been reported in China and other countries in recent years and mass mortality in abalones causes significant economic losses. For this reason, a large scale screening of immune-related abalone genes is necessary. A better unders...学位：理学博士院系专业：海洋与环境学院环境科学与工程系_环境科学学号：B20043400

Progresses on immune-related genes and proteins of abalones

鲍是世界重要的海水养殖经济贝类,但近年来时常发生的疾病给养鲍业带来了大量的经济损失。目前有关鲍免疫相关基因和蛋白的研究报道较少,而这些功能分子对揭示鲍的免疫机制具有关键作用,文章综述了鲍免疫相关基因和蛋白的研究进展,以期为防治鲍疾病的相关研究提供参考。Abalones, belonging to one of the largest marine gastropod mollusks, are economically important seafood in aquaculture worldwide.In recent years, bacterial epidemic infection has been reported in China and other countries, and mass mortality in abalones causes significant economic losses.Immune-related genes and proteins of abalones are seldom reported.However, these functional molecules may play a key role in resisting diseases and maintaining healthy status and are pivotal for studying immunological mechanisms.Here we summarized the advanced research and progresses in abalone immune-related genes and proteins with the purpose of facilitating future study of these target molecules involved in immunological mechanisms.国家高技术研究发展计划项目(863计划)(编号:2007AA091406)资

基于双通道三维效果宿主图像半色调全息水印

随着现代社会对信息承载量的需求越来越多,提出了一种基于双通道的三维效果宿主图像的半色调全息水印方法。该方法利用双通道技术将两幅三维效果宿主图像的半色调全息水印图像合成一幅图,接着制作成计算全息图,在重现的时候,一幅计算全息图能够显示出两幅水印图像。制作成的计算全息图可以增加信息传输中的安全性与保密性,双通道技术可以增加传输过程中的信息量,三维效果技术可以在不改变编码效率的情况下增强视觉效果。半色调编码全息水印能更好地提升全息水印的鲁棒性。厦门大学校长基金(中央高校基本科研业务费专项资金)资助(20720160016

Cloning and sequence analysis of hepcidin-like cDNA Hepc2 from liver of Lateolabrax japonicus

Hepcidin(Hepc)是一类性质独特的抗菌肽,具有广谱的抗革兰氏阳性、阴性细菌和真菌等作用。利用RT-PCR和RACE等技术,从经过多种细菌攻毒的花鲈(Lateolabrax japonicus)肝组织中克隆出Hepc全长cDNA,命名为Hepc2,Genbank登录号为AY604195。Hepc2有581个碱基,其中阅读框有258个碱基,编码86个氨基酸。预测氨基酸序列和白鲈及其他鱼种在相同保守的区域有8个半胱氨酸,相对分子量为9418.55dal。在3’非编码区有225bp,包含终止密码子下游189nt处的多腺苷酸化AATAAA信号和212nt处的polyA信号。预测蛋白的信号肽断裂位点在第24和第25个密码子之间。通过与白鲈(Morone chrysops)、人和其他鱼种来源的Hepc cDNA和蛋白质同源性分析表明,从花鲈肝中分离的Hepc2 cDNA属于Hepc基因家族的新成员。Hepcidin is a unique antimicrobial peptide which exerts broad-spectrum antimicrobial action against Gram-negative and Gram-positive bacteria,as well as fungi.A Hepc-like cDNA was amplified from the liver of Lateolabrax japonicus challenged with a mixed bacteria solution.Using RT-PCR and RACE with a specific primer pair,a full length cDNA sequence Hepc2 of the Hepc-like antimicrobial peptide(GenBank accession number:AY604195) was obtained.Hepc2 cDNA is composed of 581 bases,which contains an ORF of 258 bases,encoding 86 amino acids.The deduced amino acid sequence is conserved between white bass and other fish species,which share eight cysteines at the identical conserved position.The relative molecular weight of the protein is 9418.55dal.The 3′non-coding region is composed of 225bp,with a polyadenylation signal AATAAA sequence appearing at position 189 nt,and poly(A) tail at 212 nt,downstream codon TAA.The signal peptide cleavage site of its deduced protein is presumed between codon 24 and 25.High homologies with Hepc cDNAs and proteins of white bass(Morone chrysops),human and other fish are shown.It indicates that Hepc2 cDNA from Lateolabrax japonicus liver is a new member of the Hepc gene family.福建省重大科技项目(2003I005);; 厦门市高新技术项目(3502Z20021052)资

一种实验用的甲壳类生物养殖装置

一种实验用的甲壳类生物养殖装置，包括缸体，缸体由锥形底部和圆柱主体组成，锥形底部和圆柱主体之间用活动底板隔开，活动底板上设有多个过滤孔，所述活动底板上过圆心、沿竖直方向至少设有一块隔板，隔板上左右水平对称开有多排通孔，所述圆柱主体上部连接有进水管，进水管左右管壁上设有多个出水孔，圆柱主体下部外设有出水龙头。本实用新型提供的一种实验用的甲壳类生物养殖装置，通过在缸体内设置活动底板、隔板、进水管及出水龙头，一方面为甲壳类生物提供了一个良好的生活环境，另一方面由于该装置部分结构可拆卸，可同时为鱼类养殖实验所用，提高了实验装置的利用率和灵活性。</p

Construction of SSH library from haemocyte of variously colored abalone challenged with bacteria and differential expression analysis of macrophage expressed protein

以雌性杂色鲍为对象,以大肠杆菌、副溶血弧菌、溶壁微球菌、表皮葡萄球菌、金黄色葡萄球菌的混悬液做为攻毒菌,利用抑制性差减杂交(SSH)技术构建细菌攻毒的杂色鲍血淋巴细胞SSHcDNA文库。随机挑取生长菌落110个克隆子,进行菌液PCR鉴定,计算文库重组率为98.18%,文库容量为1.37×106pfu。将重组子测序,经BLAST一致性搜索比对分析,有一重组片段含有穿孔素(Perforin)保守结构域,为巨噬细胞表达蛋白(MEP)类穿孔素部分cDNA序列,片段大小为1551bp,连续编码517个氨基酸残基,申请GenBank登录号为EU272049。经半定量PCR和荧光定量PCR差异显示分析,发现在细菌感染状态下MEP基因在血淋巴细胞中存在明显的上调表达现象。Abalones are considered to be the most precious delicacy from the sea, and become very important commercial seafood in aquaculture worldwide. Variously colored abalone (Haliotis diversicolor Reeve, 1846) has been widely cultured on the southeast coast for more than twenty years. However, abalone culture frequently suffers from bacterial infection and mass mortality of reared abalones causes serious economic losses. Unfortunately, knowledge of the defense mechanism in this animal is still lacking. In this study, using suppression subtractive hybridization (SSH) technology, a forward SSH li-brary was constructed from haemocytes of H. diversicolor, with the content of 1.37×106 pfu and the recombinant rate of98.18%. After the recombinant plasmids were sequenced, partial cDNA of macrophage expressed protein (MEP) was recognized based on BLAST searches in NCBI, with the size of 1 551 bp, and continuously encoding 517 amino acids. Semi-quantitative PCR and quantitative real-time PCR results showed that MEP cDNA was distinctly up-regulated in haemocytes of the bacterial-challenged group compared to the unchallenged group. The gene information obtained from this library will provide new insights into the immune mechanism of H. diversicolor and facilitate future study of target genes involved in the response to invading microorganisms.国家高技术研究发展计划项目(863计划)(编号:2007AA091406)资助~

脆性组氨酸三联体基因蛋白表达与炎症性肠病的临床关系

目的:探讨脆性组氨酸三联体基因(fragile histidine trail, FHIT)蛋白在38例溃疡性结肠炎(ulcerative colitis,UC)、25例Crohn’s病、11例UC相关性腺癌和13例正常对照组中的表达情况. 方法:标本常规石蜡包埋,采用免疫组化法检测FHIT在UC、Crohn’s病、UC相关性腺癌和正常对照组中的表达. 结果:38例UC中,25例FHIT蛋白表达阴性;25例Crohn’S 病中,19例表达阴性;11例UC相关性腺癌中,6例表达阴性.FHIT蛋白在UC和正常对照组(71.43%vs 7.69%, X2=18.80,P0.05). 结论:FHIT蛋白在炎症性肠病(inflammatory bowel disease, IBD)和UC相关性腺癌中呈低表达或缺失,提示该基因可能与IBD和UC相关性腺癌发生有关

The Effects of Benzo [a] Pyrene (BaP) Exposure on the CYP1A1 mRNA and AhR2 mRNA Expression of Red Seabream(Pagrus major)

通过水体暴露方式对海水养殖真鲷进行bAP持续染毒,利用实时定量PCr技术研究了真鲷细胞色素P450基因(CyP1A1)和芳香烃受体基因(AHr2)随bAP暴露剂量、时间的动力学变化。结果发现,0.1~1.5μg/l环境浓度的bAP能够显著性诱导CyP1A1基因和AHr2基因的表达,且AHr2 MrnA早于CyP1A1 MrnA被诱导表达;bAP持续暴露48 H,CyP1A1和AHr2基因的表达水平均随暴露时间的延长而显著升高,染毒72 H后又回复到本底水平,实验表明这两个基因的表达与bAP的暴露剂量和暴露时间之间具有显著性的剂量-效应和时间-效应关系。The gene expression patterns of CYP1A1 and Aryl hydrocarbon receptor(AhR2) of red seabream(Pagrus major) were both measured using real-time quantitative PCR(qPCR) when fish exposed to environmentally relevant concentration of BaP(0.1,0.5 and 1.0 μg/L,respectively).The results showed that CYP1A1 mRNA and AhR2 mRNA could be induced significantly,besides,the time of AhR2 mRNA induced ahead of the time of CYP1A mRNA induced.The two genes were induced markedly at the begining of BaP exposure,and then decreased to the basal levels after 72 h.The results demonstrate that BaP can regulate CYP1A1 and AhR2 transcript in a dose and time dependent manner.国家“863”计划重点资助项目(2007AA091406);国家自然科学基金资助项目(30770391

STUDY ON THE CONSTRUCTION AND IMMUNOLOGY CHARACTERISITCS of SIX-VA-LENT FUSION TOXIN of FOOD POISONING-BORNE BACTERIA

[目的]构建4种食源性致病菌融合毒素基因及重组表达载体,制备六联融合毒素的血清抗体。[方法]采用柔性lInkEr序列(g-g-g-g-S)对目的基因进行串联(HblA-VT1b-SEA-VT2b-bOnTAHC-SEb),构建重组表达质粒PET-22b(+)-f6并在E.COlI bl21中进行表达,将表达蛋白纯化后免疫豚鼠制备血清抗体,利用ElISA和琼脂扩散试验验证抗体的特异性与敏感性。[结果]成功构建了重组表达质粒PET-22b(+)-f6并在E.COlI bl21中成功表达,37℃表达蛋白主要以包涵体形式存在(表达量10.2%),基因序列全长3384bP,编码1127个氨基酸,蛋白分子量为127205,测序结果与设计序列一致性为100%。ElISA和琼脂扩散试验表明,融合毒素f6与4种食源性致病菌有良好的反应原性,与多种非目标菌不反应。[结论]成功构建了多联融合毒素基因的表达质粒及制备了抗血清,为利用融合毒素的方法检测食源性致病菌,进而建立食源性致病菌广谱、快速的检测方法奠定基础。[Objective] To construct toxin including fusion toxin gene from food poisoning bacteria and its recombinant expression vector,and then prepare serum antibody of Six-valent fusion toxin.[Methods] Six gene(HblA—VT1B—SEA— VT2B—BoNTaHc—SEB)fragments were connected by SOE-PCR via linker sequence encoding five amino acids(G-G-G-G-S),the recombination expression plasmid pET-22b(+)-F6 was constructed and expressed in E.coli BL21.The expression proteinum was purified,and the blood serum was prepared by the immune cobaya,The specificity and sensitivity of antibody were verified by the ELISA and agar diffusion reaction.[Results] The fusion gene F6(HblA—VT1B—SEA—VT2B—BoNTaHc—SEB)and re-combinant plasmid pET-22b(+)-F6 was successfully constructed.The most of the 37℃ expression proteinum showed to be cyt-orrhyctes(expreesion amounted to 10.2%).The sequence encoding the mature fusion protein of the F6 toxin gene was 3384bp,encoding 1127 amino acids.The molecular weight of recombinant fusion toxin protein was 127.205 ku.The result of sequencing was consistent with predicted gene sequences.The results of ELISA and agar diffusion reaction demonstrated that fusion gene F6 and other four kinds of food poisoning-born bacteria had favorable reactionogenicity and did not response to many other non-tar-get bacteria.[Conclusion] The expression plasmid of multi-valent fusion toxin was successful constructed and antiserum was prepared,which laid the foundation for establishing broad spectrum and rapid detection method for food poisoning bacteria by utilization of fusion toxin detection methods.国家自然基金资助项目(30671762);厦门市科技计划项目(3502Z20055009