栉孔扇贝抗氧化酶基因的克隆与表达分析

IOCAS-IR > 海洋环流与波动重点实验室

	栉孔扇贝抗氧化酶基因的克隆与表达分析
其他题名	Molecular cloning and expression of antioxidant enzyme genes from Zhikong Scallop Chlamys farreri
	倪多娇
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	栉孔扇贝基因克隆实时定量pcr 抗氧化酶
摘要	扇贝是我国海水养殖的重要品种，但自1994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，而且直接威胁到现有产业的生存和发展。扇贝病害的不断爆发以及病因的多样性迫切要求制定新的疾病防治措施和开发新型的抗菌物质。因此，深入研究扇贝免疫防御机制，探讨提高机体抗病力的有效途径和方法，改良种质和培育抗病品系，无疑是解决目前困扰扇贝养殖业健康可持续发展的必经之路。抗氧化酶可以清除活性氧，是维持机体内氧环境平衡，抵抗外界环境影响的重要免疫因子。本研究采用大规模 EST 测序方法和同源克隆的方法，结合 cDNA 末端快速扩增（RACE）技术，从栉孔扇贝中克隆到了超氧化物岐化酶(Superoxide dismutase，SOD)、过氧化氢酶(Catalase，CAT)、谷胱甘肽过氧化物酶（Glutathione peroxidase，GPX）等抗氧化酶基因的全长 cDNA序列，并对其基因结构进行了分析。同时，用实时定量PCR方法对这三个基因在健康扇贝血淋巴细胞、肾、鳃、肌肉、性腺等组织和在分别用鳗弧菌，溶壁微球菌和假丝酵母处理扇贝后不同时间段的表达差异情况进行了研究。超氧化物歧化酶基因CfSOD的cDNA 全长为1022 bp，其中开放阅读框（Open Reading Frame, ORF）含有 459 bp，编码 153个氨基酸残基，无信号肽，为胞内蛋白。经BLASTP分析发现，CfSOD与其它动物具有较高的同源性。CfSOD中存在两个Cu/Zn-SOD的签名序列；另外Cu结合必须氨基酸(His-45，-47，-62 和-119)和Zn结合必须氨基酸(His-62，-70，-79和Asp-82)在CfSOD中保守。实时定量PCR 检测发现，CfSOD在鳃、血细胞和肾中有较高的表达。在鳗弧菌和溶壁微球菌刺激后，CfSOD的相对表达量逐渐下降，然后分别在32小时和16小时的时候恢复到刺激前的表达水平。在假丝酵母刺激后，CfSOD的mRNA表达没有显著差异。栉孔扇贝过氧化氢酶基因CfCAT的cDNA全长为3146 bp，其中开放阅读框含有1521bp，编码507个氨基酸残基，无信号肽，为胞内蛋白。经BLASTP分析发现，CfCAT与其它动物具有较高的同源性。 CfCAT中存在过氧化氢酶近端活性位点和过氧化氢酶近端血红素配体签名序列，另外存在两个糖基化位点 NFS和 NFT，同时在CfCAT 的C末端存在过氧化物酶体定位信号AQL，为典型过氧化氢酶。实时定量PCR 检测发现，健康的扇贝中CfCAT在鳃和性腺中有较高的表达。CfCAT基因在鳗弧菌刺后表达升高，在4小时达到最高，约是刺激前表达量的6.8倍（P<0.05），后随着时间的变化而逐渐下降。在8小时表达量达到为刺激前表达量的1.3倍（P<0.05），在16和32小时略高于刺激前的水平。在溶壁微球菌刺激后CfCAT基因表达量也呈上升趋势，在刺激后4小时达到刺激前表达量的约2倍，然后有所下降，在16 小时又上升到刺激前表达量的2.9倍。CfCAT基因在假丝酵母刺激后的表达略有升高，4小时约是刺激前的1.2倍（P<0.05），在其他时间段变化不明显。栉孔扇贝谷胱甘肽过氧化物酶基因CfGPX的cDNA 全长为1290 bp，其中开放阅读框含有705bp，编码235个氨基酸残基，有一个24核苷酸的信号肽序列。经BLASTP 分析发现，CfSOD与其它动物具有较高的同源性。CfGPX中发现谷胱甘肽过氧化物酶活性位点的签名序列，另外发现硒半胱氨酸和硒半胱氨酸插入序列，为含硒型谷胱甘肽过氧化物酶。实时定量PCR 检测发现，未经处理的扇贝中CfGPX在性腺、肌肉、血和肾中有较高的表达。CfGPX基因在鳗弧菌刺后表达量快速上升，在6 小时的时候表达量达到最高，为刺激前的4.0倍（P>0.05），后随着时间的变化而逐渐下降。在溶壁微球菌刺激后CfGPX在前6小时表达略有降低，在6小时的时候表达量为刺激前的0.5倍（P<0.05），后随着时间的变化而逐渐升高。在16小时的时候表达量为刺激前的2.1倍（P<0.05）。在假丝酵母刺激后， CfGPX的表达量略有下降在8小时的时候表达量为刺激前的0.8倍（P<0.05）。实验证明栉孔扇贝的超氧化物歧化酶基因CfSOD，过氧化氢酶基因CfCAT，谷胱甘肽过氧化物酶基因CfGPX基因在机体抵抗外界微生物刺激中起到了重要的作用。
其他摘要	Zhikong scallop Chlamys farreri is a commercially important cultrued species in China. The scallop industry has grown rapidly over the last 20 years. Since the large-scale mortality of cultured scallop happened in 1994， the frequently outbreaks of diseases have resulted in declined production of farmed scallop. Understanding the immunity of scallop may contribute to develop strategies for management of diseases and for long-term sustainability of scallop culture. Antioxidant enzymes that can remove reactive oxygen species and reduce oxidative stress play important role in immune system. In present study, large scale EST technique， homology-base technique and RACE technique were used to clone superoxide dismutase， catalase and glutathione peroxidase genes in Zhikong scallop C. farreri and the structures of these genes were analysized. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of CfSOD in different tissues and the temporal expression of CfSOD in scallop challenged with L. anguillarum, M. luteus and C. lipolytica respectively. The full-length cDNA of CfSOD was of 1022bp with an ORF of 459bp encoding 153 amino acids without a signal peptide. BLAST analysis revealed that the predicated amino acid sequence of CfSOD shared high identity with Cu/Zn-SOD in other organisms. There were two Cu/Zn-SOD signature sequences in CfSOD， and the amino acids required for the Cu (His-45, -47, -62 and -119) and Zn (His-62, -70, -79 and Asp-82) were conserved in CfSOD. Higher-level mRNA expression of CfSOD was detected in the tissues of haemocytes, gill filaments and kidney. The expression of CfSOD was down-regulated and then recovered after L. anguillarum and M. luteus challenged, but was not induced by C. lipolytica challenge. The full-length cDNA of CfCAT was of 3146 bp with an ORF of 1521bp encoding 507 amino acids without a signal peptide. BLAST analysis revealed that the predicated amino acid sequence of CfCAT shared high identity with CAT in other organisms. There were one catalase proximal active site signature and one catalase proximal heme-ligand signature sequence in CfCAT， and the peroxisomal targeting signal AQL were conserved in CfCAT. Higher-level mRNA expression of CfCAT was detected in the tissues of gonad and gill filaments. The expression of CfCAT was induced in the first 4 hours and reached the highest point (about 6.8 fold higher than blank group) in 4 hour point then recovered after L. anguillarum challenged; CfCAT was induced in the first 16 hours and and reached the highest point (about 2.9 fold higher than blank group) in 16 hour point after M. luteus challenged; CfCAT was increased gradually, at the 4 hour point, the expression of CfCAT was about 1.2 fold higher than the blank group while had no significance in other time points after challenged by C. lipolytica. The full-length cDNA of CfGPX was of 1290 bp with an ORF of 705bp encoding 235 amino acids with a 24 amino acids signal peptide. BLAST analysis revealed that the predicated amino acid sequence of CfGPX shared high identity with GPX in other organisms. There was one GPX active signature sequence in CfGPX， and the sedieum-cystein was conserved in CfGPX. There was a SElenoCysteine Insertion Sequence in the 3’ UTR of CfGPX. Higher-level mRNA expression of CfGPX was detected in the tissues of gonad and muscle. The expression of CfGPX was induced in the first 6 hours and reached the highest point (about 4.0 fold higher than blank group) in 6 hour point then recovered after L. anguillarum challenged; CfGPX was decreased in first 6 hours, and then increased after that in M. luteus challenged group; CfGPX was decreased a little after C. lipolytica challenged. The results indicated that CfSOD, CfCAT and CfGPX were constitutive and inducible acute-phase proteins and could play an important role in the immune responses against microorganisms infection.
页数	136
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1147
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	倪多娇. 栉孔扇贝抗氧化酶基因的克隆与表达分析[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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