Comparison of two highly expressed antimicrobial peptides Scygonadin and SCY2 in Scylla paramamosain gonads and their mechanism of reproductive immunology

拟穴青蟹(Scyllaparamamosain，以下简称青蟹)是我国南方最重要的海水养殖蟹类品种。近些年来，随着青蟹养殖规模的不断扩大，各种病害频繁发生引起养殖青蟹大量死亡，严重影响了我国青蟹养殖业的发展。青蟹以先天性免疫为主，抗菌肽是先天性免疫系统中重要免疫因子，对抵抗病原微生物感染发挥关键作用。 Scygonadin(SCY1)和SCY2是本实验室从雄性拟穴青蟹射精管中发现的两种新抗菌肽基因，前期研究已分别阐明了两种抗菌肽的基因组成与部分特性，并证明SCY1具有生殖免疫功能。青蟹半开放的生殖系统使得生殖道长期暴露于潜在病原微生物的环境中，因此研究该类抗菌肽与生殖免疫的关系对于揭示海水养殖...The mud crab, Scylla paramamosain is the most important marine breeding crab in northern China. In recent years, the farming of this species has grown rapidly. However, this economic species frequently suffered from outbreaks of disease which had led to decrease in production and severe economic loss. Antimicrobial peptides are major component of the innate immune defense system in S. paramamosain...学位：理学博士院系专业：环境与生态学院_环境科学学号：2262009015327

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~

皱纹盘鲍内脏脂质对人肝癌细胞HepG2脂质调节作用研究

本研究探讨了皱纹盘鲍(Haliotis discus hannai)内脏脂质(Haliotis discus hannai visceral lipid,HDHL)对人肝癌细胞HepG2的脂质代谢的影响,对HDHL脂肪酸成分、HepG2细胞活性、细胞内胆固醇和甘油三酯含量以及脂肪...海洋公益性行业科研专项(201405016

皱纹盘鲍内脏脂质对人肝癌细胞HepG2脂质调节作用研究

本研究探讨了皱纹盘鲍(Haliotis discus hannai)内脏脂质(Haliotis discus hannai visceral lipid,HDHL)对人肝癌细胞HepG2的脂质代谢的影响,对HDHL脂肪酸成分、HepG2细胞活性、细胞内胆固醇和甘油三酯含量以及脂肪酸代谢相关mRNA基因的表达情况进行了研究。研究发现HDHL中不饱和脂肪酸占总脂肪酸含量59.50%,其中多不饱和脂肪酸占29.71%。HDHL与HepG2细胞共孵培养24 h及48 h后,浓度为0～240μg/mL的HDHL对HepG2细胞无毒性影响。30～240μg/mL HDHL可显著降低HepG2总胆固醇含量,30～120μg/mL HDHL共孵HepG2细胞后,细胞内甘油三酯含量显著降低。qPCR结果显示HDHL可显著降低HepG2细胞脂肪酸合成相关基因SREBP1c、ACC1、FAS和脂肪酸转运吸收相关基因CD36 mRNA水平的表达,提高线粒体脂肪酸氧化基因CPT1的表达。因此,HDHL可能通过抑制脂肪酸合成和脂肪酸转运,增强线粒体中脂肪酸氧化等途径有效调节细胞脂质代谢,该研究为脂质调节功能食品的开发提供理论依据。海洋公益性行业科研专项(201405016

皱纹盘鲍HdhTPX2基因在毕赤酵母中的表达及抗氧化活性研究

为研究皱纹盘鲍Haliotis discus hannai Ino硫氧还蛋白过氧化物酶（TPX2）蛋白的抗氧化活性,将HdhTPX2基因克隆至pPIC9K载体,通过电击转化至毕赤酵母GS115菌株中,获得重组表达质粒pPIC9K-HdhTPX2,经甲醇诱导及亲和层析纯化得到重组HdhTPX2蛋白,并进行质谱鉴定及体外抗氧化功能检测。结果表明:本研究中成功构建了重组毕赤酵母菌株GS115/pPIC9K-HdhTPX2,经表达条件的优化,在pH为7的培养基中用0.5%甲醇诱导表达72 h,分泌表达上清液中得到相对分子质量约为25 000的稳定表达产物,纯化后的蛋白经质谱鉴定为目的蛋白;体外活性测定发现,该蛋白清除羟自由基（·OH）能力强于维生素C,并对H2O2引起的细胞损伤具有一定的保护作用。研究表明,皱纹盘鲍硫氧还蛋白过氧化物酶HdhTPX2在毕赤酵母表达系统中得到高效表达,重组表达产物具有抗氧化活性功能。福建省自然科学基金资助项目(2017J01041);;\n国家海洋公益性行业科研专项(201405016);;\n福建省海洋生物增养殖与高值化利用重点实验室开放课题(2015fjscq04

Antioxidant enzymes from the crab Scylla paramamosain: Gene cloning and gene/protein expression profiles against LPS challenge

Recent studies revealed that antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. Catalase is one of these key enzymes which has been characterized and highly conserved from invertebrates to vertebrates. In the present study, a full-length cDNA sequence of catalase was cloned from the hemocyte suppression subtractive hybridization library of the crab Scylla paramamosain. The Sp-catalase (Sp-CAT) cDNA sequence contained 2551 bp with an open reading frame of 1551 bp encoding 517 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of Sp-CAT. The deduced Sp-CAT protein had a calculated molecular mass of 59 kDa with an estimated isoelectric point of 6.4. Multiple alignment analysis revealed that the deduced amino acid sequence of Sp-CAT shared high identity (75.4%) with those of other species. The Sp-CAT mRNA transcripts were demonstrated in multiple tissues of normal S. paramamosain. After LPS challenge, the expression level of Sp-CAT gene was increased significantly in hemocyte at 3 and 6 h, and in hepatopancreas at 6 h, respectively, determined by quantitative real-time PCR. Furthermore, the activities of CAT and SOD were also measured in different tissues and serum after LPS challenge. The CAT activity was significantly increased at 3, 6, 24 and 48 h in hemocyte lysate, at 3 h in serum, and at 24 and 48 h in hepatopancreas after LPS challenge. In addition, the SOD activity was significantly induced at 3 and 6 h in hemocyte lysate, 3 and 12 h in serum, 12 and 48 h in hepatopancreas post LPS stimulation, indicating a tissue and time-dependent antioxidant response in the crab. Taken together, these data demonstrated that a strong antioxidant response occurred in the LPS-challenged crab, which might be involved in the protection of host against microbial infections. (C) 2010 Elsevier Ltd. All rights reserved.National Natural Science Foundation of China (NSFC) [40676083]; National High Technology Research and Development Program of China [2007AA091406]; Minjiang Scholars Program [2009]; Natural Science Foundation of Fujian Province China (NSFC-FPC) [2009J05084

The Optimization of Primary Hemocyte Culture of Scylla paramamosain

本研究通过对拟穴青蟹血细胞原代培养条件的摸索与优化,成功在体外培养了青蟹血细胞。结果表明,PH 4.6的海洋甲壳类血细胞抗凝剂培养青蟹血细胞的形态优于海洋蟹类抗凝剂(ACd);高血清浓度(>15%)培养血细胞形态较差,易出现空泡;添加5%fbS与1.2%nACl的l15培养基是培养拟穴青蟹血细胞最适培养基,并可在培养的青蟹原代血细胞中检测到稳定的管家基因表达,表明该细胞可用于拟穴青蟹体外功能试验研究。The optimization of primary hemocyte culture of Scylla paramamosain was successfully studied in vitro.The results showed that the hemocyte cells were morphologically kept activity better at pH 4.6marine crustaceans anticoagulant.High concentration of fetal bovine serum(>15%)in the culture lead to development of vacuoles.The hemocyte cells survived best in L15 medium supplemented with 5%fetal bovine serum and 1.2%sodium chloride.The house keeping gene β-actin of Scylla paramamosain of cultured hemocyte maintained stably expressed through the experiment.This cell culture system could be used for in vitro experiments.福建省农业科技重点项目(2012N0029); 中国博士后科学基金面上资助(2014M550354

Molecular characterization and promoter analysis of crustacean heat shock protein 10 in Scylla paramemosain

National Natural Science Foundation of China [U1205123]; Public Science and Technology Research Funds for Ocean Projects, State Oceanic Administration of the People's Republic of China [201105027]Heat shock proteins (Hsps) are an evolutionarily conserved group of molecules present in all eukaryotic and prokaryotic organisms. Hsp10 and Hsp60 were originally described as the essential mitochondrial proteins involved in protein folding. Recent studies demonstrate that Hsp10 has additional roles including immune modulation. In our study, an homologous Hsp10 (Sp-Hsp10) was identified in the mud crab Scylla paramemosain, and its genomic DNA organization was determined. The cDNA sequence of Sp-Hsp10 contains an open reading frame of 309 bp, encoding a putative protein of 102 amino acid residues with approximately 10 kDa. The Sp-Hsp10 gene is located next to the Sp-Hsp60 gene and shares a 1916-bp intergenic region. The promoter activity of the Sp-Hsp10 flanking gene was analyzed using luciferase reporter assays in transfected endothelial progenitor cells. The upregulation of Sp-Hsp10 expression was detected after exposure of hemocytes to a heat shock of 1 h at 37 degrees C compared with unstressed hemocytes raised at 20 degrees C. To our knowledge, this is the first report characterizing the genomic organization of a new Hsp10 in a crustacean

Gene cloning of a sigma class glutathione S-transferase from abalone (Haliotis diversicolor) and expression analysis upon bacterial challenge

National High Technology Research, Development Program of China [2007AA091406]; MEL Young Scientist Visiting Scholarship [MEL0905]Glutathione S-transferases (GSTs) are a multigene family of xenobiotic metabolizing phase II detoxification enzymes which take part in many pathological and physiological processes, and which can potentially be used as indicators and biomarkers for cancer diagnoses and organic or inorganic pollutant exposure. In this study, a full-length cDNA of a sigma class GST (abGSTsigma) (CenBank accession number EF546619) from variously colored abalone (Hatiotis diversicolor) was identified. It was 1328 bp containing an open reading frame of 624 bp, encoding 208 amino acid residues with a predicted protein molecular weight of 23.67 kDa and an estimated pI of 5.67. Sequence analysis showed that the predicted protein sequence of abGSTsigma cDNA contained the conserved domain of the GST_N_Sigma_like (PSSM: cd03039) and GST_C_Sigma_like (PSSM: cd03192). Alignment analysis demonstrated that the abGSTsigma of H. diversicolor was in a branch position with other known class sigma GSTs from different organisms. The abGSTsigma mRNA was distributed in multiple tissues tested and was highly demonstrated in the gill and mantle of normal abalones. In bacteria-challenged abalone, the abGSTsigma gene was significantly expressed in the hemocytes, gill, mantle and digestive gland and the total GSTs enzyme and SOD were also induced in the four tissues. The increased activities of SOD and GSTs can result in the elimination of reactive oxygen species (ROS) indicating antioxidant activities involved. The preliminary work revealed that the sigma class glutathione S-transferase gene abGSTsigma, a phase 11 detoxification enzyme, had a positive response to bacterial challenge, and that will lead to an insightful study on elucidating the interactions between immune responses and biotransformation exerted by abGSTsigma. (C) 2009 Elsevier Ltd. All rights reserved