栉孔扇贝核心组蛋白的基因结构及H2A抗菌活性的研究

IOCAS-IR > 海洋环流与波动重点实验室

	栉孔扇贝核心组蛋白的基因结构及H2A抗菌活性的研究
其他题名	Preliminary study on the core histone gene cluster and the antibacterial activity of histone H2A from Chlamys farreri
	李成华
学位类型	博士
	2007-06-08
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	栉孔扇贝组蛋白h2a 基因克隆重组表达抗菌活性
摘要	栉孔扇贝是我国传统的海水养殖品种，但自1997 年以来，养殖扇贝陆续爆发的大规模死亡，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，虽然针对扇贝养殖环境、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。但由于引起养殖扇贝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，贝类养殖业要摆脱病害的困扰，必须开辟新的疾病防治途径。从扇贝自身的免疫防御因子入手，筛选和克隆参与免疫防御的功能基因，尤其是一些新颖的具有抗菌活性的分子，对于深入探讨扇贝的免疫防御机制，指导扇贝的遗传改良和抗病品系的培育具有重要的意义；另一方面，可对抗菌效应物实现重组表达，开发新型的病害预防治疗制剂，取代目前普遍使用的抗生素和化学药物。抗菌效应物是机体在免疫应答过程中产生的多肽类物质，对侵入生物体内的细菌、病毒具有很强的免疫杀灭作用，对抗菌效应物的研究有助于深入了解机体先天性免疫防御的机制。本研究在同源克隆策略的基础上，从利用构建的Genome Walking 文库中克隆到了栉孔扇贝核心组蛋白群的全长序列，该串联重复序列全长5671bp，包括各一个拷贝的组蛋白H4, H2B, H2A 和 H3。所有的核心组蛋白在3’侧翼序列均具有与其在细胞周期进化模式相关的特征结构，即两个不同的终止信号：发卡结构和至少一个多聚腺苷酸信号序列（AATAAA）。在5’区域的起始密码子上游37–45 bp处的保守的CAP位点（5’-PyCATTCPu-3’）存在于除H2B外的每一个基因中；规则的TATA 和CAAT元件也在核心组蛋白群中的个别的基因中找到。在H2B 和H2A基因的启动子区域，对于定位转录起始位点非常重要的元件（5’-GATCC-3’）也相对保守；在H2B启动子区域存在着与其特征序列（5’-GGAATAAACGTATTC-3’）相似性很高的序列结构5’-GGATCGAAACGTTC-3’。增强子序列只发现存在于H4 和 H3基因中，其序列结构与组蛋白增强子序列(5’-TGATATATG-3’)基本匹配。在组蛋白基因群中存在着一些保守的序列和重复结构表明组蛋白基因的进化是采取“生与死的进化模式”并伴随着强的纯化选择压力，使得该基因群变异较少以保持其基本功能。同时，利用18S rRNA做参照，探讨了H2A 和H2B作为分子系统进化分析的潜在分子标记，表明组蛋白H2A 和H2B可以作为分子系统进化分析得候选分子，它们在区分近缘种的分辨率上表现出了更高的灵敏度。该研究结果为进一步定性软体动物组蛋白重复单位提供了基础。在脊椎动物中，组蛋白H2A通过特异性剪切其N末端产生新颖的抗菌肽的形式来参与宿主的免疫应答反应，在软体动物中是否存在同样的机制还未有研究报道。本研究利用上述克隆的H2A基因研究了其在病原胁迫下的表达变化规律并对其N末端39aa进行了重组表达和抗菌活性分析，以期为开发和利用软体动物的新颖的抗菌肽提供理论依据。半定量RT-PCR发现血细胞中H2A 的mRNA 在微生物感染前后的表达量没有任何显著的变化，表明H2A本身并不直接参与对病原的清除过程或者说病原微生物并不能诱导H2A的表达。因此，我们推测该基因可能象脊椎动物一样以前体形式存在，经剪切后参与宿主的免疫应答过程，为此我们研究了H2A的N末端的抗菌活性。通过将与脊椎动物buforin I同源的H2A的N末端39aa克隆到毕赤酵母表达载体pPIC9K实现了该基因N末端的重组表达。抑菌实验表明，重组产物具有广谱的抗菌活性，其对供试的革兰氏阳性菌藤黄微球菌表现出显著的抗菌活性，而对革兰氏阴性菌（鳗弧菌、亮弧菌）的抑菌活性则相对较弱；此外，重组产物对毕赤酵母GS115也表现出一定的杀菌活性，证明其具有抗真菌活性。上述研究结果证明组蛋白H2A的N末端是一种潜在的抗菌肽，但该抗菌肽是否参与机体的免疫应答过程需要进一步的深入研究。
其他摘要	Zhikong scallop is the major specie in aquaculture and contributes enormously to the economic development of coastal provinces in China. Since the summer of 1997, large-scale mortality of cultured scallop has caused catastrophic losses to scallop aquaculture, resulting in the production decreasing drastically. The durative outbreak of diseases has accelerated intensive efforts for the development of better health management strategies and characterization of original immune efforts for disease control. Although it is still not very clear about the pathogens for the scallop mortality, the immune response of scallop to the pathogen infection plays a key role in controlling outbreak of disease. Understanding the immune defense mechanisms of scallop may contribute to develop strategies for management of the disease and for long-term sustainability of scallop or mollusk farming. The identification and characterization of genes involved in scallop immune responses are now considered to be essential for the elucidation of immune defense mechanisms and disease control because of their potential use as therapeutic agents and genetic improvement biomarkers on disease-resistant strain selection. Antimicrobial effectors constitute the first line of innate immunity for scallop exposed to various potential pathogens in the aquatic environment by exerting broad-spectrum microbicidal activity. The nucleotide sequence of the core histone gene cluster from scallop Chlamys farreri was of 5671 bp, which contained a copy of the four core histone genes H4, H2B, H2A and H3 amplified and identified by the techniques of homology cloning and genomic DNA walking. In the 3’ flanking region of all the histone genes in the cluster had the structures in their 3’ flanking region which related to the evolution of histone gene expression patterns throughout the cell cycle, including two different termination signals, the hairpin structure and at least one AATAAA polyadenylation signal. In their 5’ region, the transcription initiation sites with a conserved sequence of 5’-PyCATTCPu-3’ known as the CAP site were present in all genes except to H2B, generally 37–45 bp upstream of the start code. Canonical TATA and CAAT boxes were identified only in certain histone genes. In the case of the promoters of H2B and H2A genes, there was a 5’-GATCC-3’ element, which had been found to be essential to start transcription at the appropriate site. After this element, in the promoter of H2B, there was another sequence, 5’-GGATCGAAACGTTC-3’, which was similar to the consensus sequence of 5’-GGAATAAACGTATTC-3’ corresponding to the H2B-specific promoter element. The presence of enhancer sequences was identified from the H4 and H3 genes, matching perfectly with the consensus sequence defined for histone genes (5’-TGATATATG-3’). There were several slightly more complex repetitive DNA in the intergene regions. The presence of the series of conserved sequences and reiterated sequences was consistent with the view that mollusc histone gene cluster arose by duplicating of an ancestral precursor histone gene, the birth-and-death evolution model with strong purifying selection enabled the histone cluster less variation and more conserved function. Meanwhile, the H2A and the H2B were demonstrated to be potential good marks for phylogenetic analysis. All the results will be contributed to the characterization of repeating histone gene families in mollusc. Histone H2A is reported to participate in host-defense response through producing novel AMPs from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not been reported in mollusca to our knowledge. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate if the similar mechanism existed in mollusca. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not up-regulated after bacterial stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117 bp encoding 39 amino acids corresponding to the N terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His+ Mut+) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G+) and Gram-negative (G-) bacteria. The antibacterial activity toward G+ bacteria was two and a half times more than that against G- bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. But if H2A was really involved in scallop immune response mechanisms needed to be further investigated.
页数	124
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/861
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	李成华. 栉孔扇贝核心组蛋白的基因结构及H2A抗菌活性的研究[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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