【作者】 邢士超；

【导师】 杨官品； 陈松林；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 大菱鲆是我国重要的海水养殖鱼类,但是随着养殖环境的恶化,鱼病不断发生,不但造成了巨大的经济损失,而且严重影响了该产业的健康发展。目前,虽然针对大菱鲆养殖环境、病原以及养殖技术等方面开展了大量的研究工作,提出了许多防病治病的措施,并取得了一定的成效。但由于引起养殖大菱鲆病害的病原和发病原因的多样性,大量使用抗菌素和农药后造成病原微生物抗药性的提高以及对环境造成的严重破坏,大菱鲆养殖业要摆脱病害的困扰,必须开辟新的疾病防治途径。抗菌肽是一类具有广谱抗菌活性的小分子物质,是生物体天然免疫反应系统的重要成员,在生物体抵抗外界病原物感染过程中起着非常重要的作用。海水养殖鱼类抗菌肽重组表达为海水鱼类抗病分子育种和疾病控制提供了一条新的途径。而且,胰岛素样生长因子-I( insulin-likeg rowthf actor-1,IGF-I)是一种具有促进多种细胞生长功能的活性多肤,它对织织细胞的增殖、分化、凋亡,机体的生长发育及肿瘤的发生发展起重要的调节作用。同时,在毕赤酵母里表达抗菌肽的研究为开发转基因酵母作为鱼类饵料添加剂的应用研究提供了理论基础。鉴于此,我们开展了以下研究工作:1.克隆了大菱鲆IGF-I,荧光实时定量PCR检测表明:IGF-I普遍存在于各组织中,但表达量不同,在肝脏中最高;对大菱鲆胚胎发育早期研究表明,随着发育的延续直到孵化期,IGF-Ⅰ基因的表达呈逐渐增加的趋势;高温刺激研究表明,高温对IGF-Ⅰ基因在肝脏中的表达整体上有抑制作用;饥饿处理显示,饥饿16天已促使IGF-Ⅰ基因在肝脏中的表达水平显著下降。2.通过基因重组技术构建了5种酵母分泌融合表达载体和5种酵母胞内融合表达载体。western blot检测表明,获得了了四种酵母分泌重组表达菌株,pPICZαA-HIP、pPICZαA-HGP、pPICZαA-HP和pPICZαA-IP。并且随着诱导时间的延长,pPICZαA-HIP和pPICZαA-HGP表达蛋白量逐渐增高,然而前9个小时未检测到目的蛋白表达。抑菌实验表明:pPICZαA-HIP、pPICZαA-HGP和pPICZαA-HP都有一定的抑菌活性;pPICZαA-HP的抑菌活性最高。通过GFP荧光检测发现,在重组酵母菌株pPICZαA-HGP内和胞外都能检测到该融合蛋白的荧光信号。3.构建了5种荧光蛋白融合细胞表达载体,eGFP-HN,eGFP-IN,eGFP-HIN, eGFP-GN和eGFP-MN。转染了大菱鲆肾脏细胞系,在eGFP-HIN, eGFP-GN和eGFP-MN转染细胞系中检测到GFP荧光信号。研究发现,这些荧光信号在这些转基因细胞系中分布不同。本研究克隆了IGF-Ⅰ基因,研究了IGF-Ⅰ的表达特性;获得了几种SMD1168重组菌株;建立大菱鲆酵母重组表达体系;获得了3种转基因细胞系,为以后开展基因功能活性研究、基因互做、新型饲料添加剂的开发等工作奠定了基础。更多还原

【Abstract】 Turbot, Scophthalmus maximus, is an important species in aquaculture and it contributes enormously to the economic development of coastal provinces in China. However, with the farming environment worsening, it had caused catastrophic losses to aquaculture. The intensive efforts had been accelerated for development of better health management strategies and characterization of original immune efforts because of the outbreak of diseases. Although it is still not very clear on the pathogen infection of the turbot, the immune mechanism plays a key role in controlling outbreak of disease in turbot. Thus, understanding the immune mechanism of turbot could contribute to develop strategies for management of the disease and provide guidance about the disease control for other marine fishes.Antimicrobial peptides (AMPs) are regarded as important components of the host innate immune system and play crucial roles in host defense against microbial invasion. Based on the expression and recombination of antimicrobial peptides genes, we can find an attractive way of fishery breeding and disease control of marine fishes. And insulin-like growth factor-I (IGF-I) is a kind of active peptide that can promote many types of cells to grow. It plays an important regulatory role in the proliferation, differentiation, cell apoptosis and tumor genesis. Moreover, recombinant expression of antimicrobial peptides in yeast provides us an opportunity to explore the recombinant strains as additive feedstuff of fishery. The study mainly focuses on following research contents:1. Molecular cloning and expression analysis of turbot IGF-I gene; Quantitative real time PCR (QRT-PCR) demonstrated that IGF-I transcripts were highly abundant in liver, abundant in eye, skin, brain, gill, gonad, spleen; less abundant in muscle, heart, intestine and head kidney. The level of IGF-I mRNA in embryos gradually increases during embryogenesis after fertilization. The high temperature stimulation research indicated that IGF-Ⅰhas the inhibitory action in liver. And Expression analysis of IGF-Ⅰin starvation turbot liver decreased significantly on 16th day.2. That five secretion expression vectors of yeast were constructed with gene recombination, and five intracellular expression vectors also constructed. With western blot, it was indicated that four recombinant yeasts, pPICZαA-HIP, pPICZαA-HGP, pPICZαA-HP and pPICZαA-IP were made. The recombinant protein was detected with western blot, and it was shown that the expression of protein of pPICZαA-HIP and pPICZαA-HGP was increasing with time by methyl alcohol induced. It was shown that there was some antibacterial activity with pPICZαA-HIP, pPICZαA-HGP and pPICZαA-HP. And the activity of pPICZαA-HP was the most. And GFP was found with pPICZαA-HGP in the yeast and around.3. That five fluorescence protein fusion expression vectors of cell were constructed, eGFP-HN, eGFP-IN, eGFP-HIN, eGFP-GN and eGFP-MN. With GFP detected, it suggested that there were 3 recombinant cell lines, eGFP-HIN, eGFP-GN and eGFP-MN, made with gene recombination. And it was shown different GFP distribution in them.In summary, the IGF-Ⅰgene was cloned, and its expression was detected with QRT-PCR. It was confirmed that several recombinant yeasts were made and the recombinant yeast expression system of turbot was established. And 3 recombinant cell lines were also made. It has laid the foundation for later study on function and action of gene, gene interation, new feed additive development and so on.更多还原