Cloning,characterization and expression of the pepsinogen C from the golden mandarin fish <Emphasis Type="Italic">Siniperca scherzeri</Emphasis> (Teleostei: Perciformes)

Pepsinogens are precursors of pepsins, which are gastric digestive proteinases that degrade food proteins into peptides. In the study reported here, the cDNA and its corresponding genomic DNA of the golden mandarin fish (Siniperca scherzeri, Perciformes) pepsinogen C (PGC) were cloned and sequenced. The golden mandarin fish PGC gene was deduced to have nine exons and eight introns, a structure similar to the PGCs of other vertebrates. The full-length cDNA was found to contain a 37-bp 5′-untranslated region, a 1,164-bp open reading frame, and a 304-bp 3′-untranslated region and the PGC protein to consist of a signal peptide, an activation segment, and a pepsin moiety. A sequence analysis revealed that pairwise sequence similarities of PGC proteins are around 70% between golden mandarin fish and other vertebrate groups, and around 90% within the fish group. A comparison of vertebrate PGC protein sequences revealed two motifs. One was in the activation segment that occurred only in the mammal and avian PGCs, suggesting that PGCs active in homeotherms (mammal and avian) have different activation mechanisms than those in poikilotherms (amphibian and fish). The second was in the pepsin moiety that occurred only in fish, suggesting the primitive position of fish among vertebrates. PGC mRNA is mainly expressed in the stomach and esophagus and at much lower levels in the skin and muscle. Taken together with results reported from other studies, the results reported here will lead to a better understanding of the molecular mechanisms of fish digestive physiology and the evolution of fish pepsinogen genes.     

黄鳝载脂蛋白A1的基因结构及表达谱分析

根据克隆的黄鳝载脂蛋白A1基因的部分序列,进行5′RACE扩增和内含子的克隆并采用荧光定量PCR分析该基因的表达谱。结果表明,该基因cDNA全长1207bp,5′UTR区域长32bp,编码1个262aa的多肽;在长1391bp的gDNA上,只发现了1个长184bp的内含子。荧光定量PCR对该基因在不同组织和病原细菌嗜水气单胞菌感染后的表达情况分析表明,该基因转录本在肝脏中表达量最高,在胃、肾脏、小肠、脑、皮肤和血液中表达量中等,而在心脏、脾脏和肌肉中表达量很低;病原细菌感染会显著影响该基因在小肠、肝脏和脾脏中的表达水平。以上结果显示黄鳝的载脂蛋白Apo-A1基因可能参与了鱼体的天然免疫反应。     

半滑舌鳎TLR5S 三种剪切型基因的克隆与表达分析

Toll样受体5(Toll-like receptor 5,TLR5)是TLRs家族成员之一,可分为跨膜型TLR5M和鱼类特有的可溶型TLR5S,它们可以识别致病菌表面的鞭毛蛋白并协同作用激活免疫反应。为了研究半滑舌鳎受到病原感染后TLR5S参与免疫反应的作用,本研究使用RACE技术获得了半滑舌鳎(Cynoglossus semilaevis)TLR5S全长c DNA序列。TLR5S c DNA有3种剪切型:Cs.TLR5S x1,Cs.TLR5S x2和Cs.TLR5S x3。这3种剪切型的相同区域为308 bp 5′非编码区(5′UTR)和1701 bp开放阅读框(ORF),不同的3′UTR分别为138 bp、364 bp和637 bp。Cs.TLR5S共编码567个氨基酸,预测编码蛋白质分子量为64.03 k D,等电点为8.49。氨基酸多重序列比对结果显示,Cs.TLR5S氨基酸序列与其他脊椎动物TLR5S氨基酸序列具有较高的相似性,其中与牙鲆相似度高达61%,表明Cs.TLR5S在进化上的具有一定的保守性。Real-time PCR结果表明该基因在半滑舌鳎的不同组织均有表达,其中在肝的表达量最高,在脾的表达量最低。此外,检测Cs.TLR5S 3′端的不同剪切型在肝、脾、头肾、小肠中的表达,结果显示Cs.TLR5S x3只在肝中高表达,而Cs.TLR5S x1则在肝和小肠中都有中等程度表达。鳗弧菌(Vibrio anguillarum)感染半滑舌鳎实验表明,注射菌液6 h后,Cs.TLR5S基因在肾、小肠、肝和脾4个组织中的表达量都有显著上升;注射鳗弧菌48 h后,以上4种组织中表达量均呈现降低的变化。上述实验结果说明,Cs.TLR5S基因可能参与了半滑舌鳎抗弧菌感染的免疫反应。     

尼罗罗非鱼p38MAPK基因克隆与表达分析

为了探究p38MAPK与尼罗罗非鱼(Oreochromis niloticus)免疫功能的相关性,本实验运用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)获得尼罗罗非鱼埃及品系ntp38MAPK的cDNA全长序列,结果显示,该序列全长1789 bp,开放阅读框(Open Reading Frame,ORF)长1086 bp,5′非编码区(Untranslated Region,UTR)长342 bp,3′UTR为361 bp。ORF编码361个氨基酸,预测分子量为41.598 kD,理论等电点为5.10。序列含有p38家族典型保守的Thr-Gly-Tyr(TGY)双磷酸化位点以及紧密相连的底物结合位点Ala-Thr-Arg-Trp(ATRW)。同源性以及系统进化树分析结果显示,尼罗罗非鱼的p38MAPK与大黄鱼(Larimichthys crocea)和鲈(Dicentrarchus labrax)的相似性最高。采用qRT-PCR的方法研究了ntp38MAPK在各组织以及无乳链球菌感染过程中的表达差异情况,结果显示,ntp38MAPK在各组织均有表达,其中在肌肉的表达量最高,心脏、肝脏、脾脏次之,头肾中的表达量最低。无乳链球菌感染过程中,脾脏、头肾中ntp38MAPK的表达量在2 h开始上调,肝脏中4 h开始上调,8 h后肝脏、脾脏和头肾中的表达量都有所下降,24~72 h趋于平稳,表明ntp38MAPK在尼罗罗非鱼抵抗链球菌侵染过程中发挥重要作用。