【作者】 谢芳靖；

【导师】 王艺磊；

【作者基本信息】 集美大学 ， 水产养殖学， 2007， 硕士

【摘要】 对大黄鱼腹腔注射副溶血弧菌(实验组)或0.9%氯化钠溶液(对照组),在注射4h、1d、2d、4d、8d、12d和16d后尾部取血,测定其血清中一氧化氮合酶、溶菌酶和酪氨酸酶活力的变化情况。结果表明,注射副溶血弧菌后大黄鱼血清中溶菌酶活力在2d和12d显著高于对照组,在16d则显著低于对照组;酪氨酸酶活力在4d和16d显著高于对照组,在8d极显著高于对照组;但大黄鱼体内的一氧化氮合酶活力没有明显增加。表明大黄鱼血清中的溶菌酶和酪氨酸酶参与了机体对副溶血弧菌的免疫防御。本研究在上述结果的基础上,利用RT-PCR技术克隆了大黄鱼TNFα、Mx、IgL和IgH基因,并采用定量PCR技术分析了这四个基因的表达情况。结果如下:(1)TNFαcDNA序列全长1339 bp,5’UTR为68 bp,ORF为759 bp,3’UTR为512 bp,可编码252个氨基酸,分子量大约为28kDa。由该序列推导的多肽含有跨膜域及TNF家族的标签序列。同源性分析表明该序列与其它硬骨鱼类甚至哺乳动物的TNFα具有很高的相似性。进化分析显示,大黄鱼TNFα与其它硬骨鱼类TNFα占据进化上独立的分支,而哺乳类TNFα则形成另一分支。定量PCR分析发现,在头肾中2d和4d的弧菌组大黄鱼TNFα基因表达极显著地高于对照组;在血液中2d的弧菌组大黄鱼TNFα基因表达显著地高于对照组,而4d的弧菌组大黄鱼TNFα基因表达极显著地高于对照组。(2)获得的2209bp Mx全长cDNA含有一个1890bp ORF,可编码629个氨基酸,分子量大约为72kDa。在可编码序列中,具有Mx蛋白的特征结构,如GTP酶基序、GTP酶动态蛋白超家族标签序列(LPRGSGIVTR)和C-端区域的亮氨酸拉链结构等。对大黄鱼腹腔注射副溶血弧菌后2d,其头肾和血液中的Mx基因表达均显著上调,且在2d均达到峰值。(3)克隆到一个977bp IgL cDNA全长序列和一个1621bp的IgH cDNA片段。IgL编码区由可变区和恒定区两个结构域组成;其互补决定区变化较大,与其它鱼类相似性较低。进化分析显示,大黄鱼与其它鱼类IgL独立类聚成一支。定量PCR显示,注射副溶血弧菌后8d和12d,大黄鱼头肾中的IgL和IgH基因表达均显著地高于对照组。引物退火控制技术(ACP)是一种在DDRT-PCR基础上发展起来的对差异表达基因进行克隆的新方法。退火控制引物3’端的核心部分和5’端的通用引物序列部分由中间的多聚脱氧次黄苷[Poly(dI)]调节部分连接,可以显著提高引物退火的特异性。该方法具有重复性高、假阳性低、快速经济和PCR产物分布范围广等特点。通过对这一方法的改良,克隆到一个大黄鱼抗病相关的1360bp纤维蛋白原β链基因片段。更多还原

【Abstract】 Serum of large yellow croaker was collected at 4h, 1d, 2d, 4d, 8d, 12d, 16d after intraperitoneal injection with Vibrio parahaemolyticus(experimental group)or 0.9% NaCl solution (control group), and the response of selected innate immune parameters (nitric oxides synthase, lysozyme and tyrosinase) was investigated. The lysozyme activities in the serum of large yellow croaker in the experimental group were significantly higher than that of the control group at 2d and 12d after injection, while at 16d after injection, it was significantly lower than that of the control group. The tyrosinase activities in the serum of large yellow croaker in the experimental group were significantly higher than that of the control group at 4d and 16d, and at 8d after injection it was much significantly higher than that of the control group. Nitric oxides synthase activities did not show statistical difference between experimental and control group during the exposure. These results indicate that lysosyme and tyrosinase play an important role in immune defence of V. parahaemolyticus in large yellow croaker.Base on the above results, RT-PCR and real time PCR were used to clone and analyse the four genes of TNFα, Mx, IgL and IgH from large yellow croaker. The main results are reported as follow: (1) The full length cDNA of TNFαcontained a 5’UTR of 68 bp, followed by an ORF of 759 bp, and a 3’UTR of 512bp. The ORF was capable of encoding 241 amino acids with an estimated molecular mass of 28 kDa. The deduced peptide had a transmembrane domain and a TNFαfamily signature. Large yellow croaker TNFαshares relatively high similarity with both teleost and mammalian TNFαby multiple sequence alignments. Phylogenetic analysis showed that the teleost TNFαwere located independently in a different branch compared with mammalian TNFα. Real time PCR analyses demonstrated that the expression of TNFαin head kidney of large yellow croaker injected with V. parahaemolyticus were much significantly higher than that of the control group at 2d and 4d; in blood, at 8d after injection it was significantly higher than that of the control group, while at 4d after injection it was much significantly higher than that of the control group. (2) The Mx full length cDNA of 2209 bp contained an ORF of 1890bp that coded for a protein of 629 amino acids with an estimated molecular mass of 72 kDa. Within the coding sequence, characteristic features of Mx proteins were found, such as a GTP-binding motif, the signature of the dynamin family (LPRGSGIVTR), and a sequence that coded for a leucine zipper at the C-terminal region of the protein. Intraperitoneal challenge of large yellow croaker with V. parahaemolyticus significantly up-regulated Mx expression in head kidney and blood at 2d, and also peaked at 2d. (3) The 977bp full length cDNA of IgL and 1621bp fragment cDNA of IgH have been cloned. IgL encoding comprising its variable (VL) and constant (CL) regions, which were conserved with their putative domains; and its exchanges were present mainly within CDRs. The phylogenetic analyses also demonstrated that large yellow croaker and other piscine IgL cluster as a separate branch out of the mammalian branches. Moreover, real time PCR revealed that the expression of IgL and IgH in head kidney large yellow croaker injected with V. parahaemolyticus were significantly higher than that of the control group at 8d and 12d, respectively.Annealing control primer (ACP) system is a novel method that is based on DDRT-PCR, which can be used to identify differentially expressed genes. The primer comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3′end target core sequence and the 5′end non-target universal sequence, resulting in a dramatic improvement of annealing specificity. It has the key features of higher reproducibility, low false positives, speed and cost-effectiveness and a wide range of PCR products. We have used this method with some modification to clone a 1360bp fragment of fibrinogen beta chain gene which response to a pathogen challenge in large yellow croaker.更多还原