海湾扇贝超氧化物歧化酶家族基因结构、表达和多态性分析

IOCAS-IR > 海洋环流与波动重点实验室

	海湾扇贝超氧化物歧化酶家族基因结构、表达和多态性分析
其他题名	Superoxide dismutase multigene family in bay scallop Argopecten irradians: gene structures, expression and polymorphism
	包永波
学位类型	博士
	2009-05-17
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	海湾扇贝超氧化物歧化酶基因结构启动子 Qrt-pcr 鳗弧菌免疫反应酶活分析 Snp
摘要	海湾扇贝Argopecten irradian Lamarck于1982年从美国引种到中国，由于具有较快的生长速度和很高的经济效益，海湾扇贝成为中国最主要的养殖贝类之一。近年来海湾扇贝养殖遇到了死亡率高等问题，深入开展海湾扇贝功能基因的研究，尤其是免疫相关基因及其机制研究并在此基础上寻找扇贝疾病防治的有效方法对海湾扇贝的健康养殖十分重要。对于贝类免疫系统来说，其血细胞在先天性免疫防御中起着重要的作用。当受到外界病原侵染时，贝类血细胞的一个重要免疫反应就是吞噬作用。在吞噬病原过程中，受到病原侵染的贝类还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发与呼吸链相耦联的活性氧（ROS）的大量产生。这些活性氧具有极强的反应特性，能破坏病原微生物的结构和功能分子，实现对入侵病原的杀灭。利用活性氧对被吞噬的病原进行杀灭，这是吞噬作用消除病原抵御侵染的重要机制。但由于活性氧分子反应的非特异性，它们也会破坏宿主机体细胞内的功能蛋白分子、不饱和脂肪酸分子和核酸等，对细胞造成严重的伤害，进而导致机体生理机能的损伤和免疫系统的破坏。所以，及时消除病原感染机体内过量产生的ROS，维持相关细胞的正常代谢，对提高机体抵抗力和免疫力具有重要的作用。O2-是生物体内产生的第一种活性氧分子，其他的活性氧分子也是由它衍生而来，消除过量O2-是消除过量活性氧危害的第一步也是关键一步。生物体内，超氧化物歧化酶（SOD）是催化O2-发生歧化反应，消除O2-的关键酶。首先，本文通过RACE方法获得了海湾扇贝SOD家族全部三种基因的cDNA全长并对其进行了序列的生物信息学分析，海湾扇贝AiCuZnSOD全长cDNA为1047个碱基，其中开放阅读框为459个碱基，编码152个氨基酸，与栉孔扇贝Chlamys farreri的CuZnSOD相似度为77.5%，与长牡蛎Crassostrea gigas的相似度为75%，与人的相似度为74.7%。AiMnSOD全长cDNA为1207个碱基，其中开放阅读框为678个碱基，编码226个氨基酸，序列比对结果发现AiMnSOD的氨基酸序列与虾夷扇贝Mizuhopecten yessoensis和皱纹盘鲍Haliotis discus hannai的相似度分别为85%和78.4%，与哺乳动物相似度也在68%~72%之间。AiECSOD全长cDNA为893个碱基，其中开放阅读框为657个碱基，编码218个氨基酸。AiECSOD与其它物种ECSOD相似度比较低。与线虫Brugia pahangi的相似度为27.9%，与疟蚊Anopheles gambiae的相似度为31.4%，与斑马鱼Danio rerio的相似度为27.8%，与人的相似度也只有28.6%，与同是贝类的长牡蛎ECSOD也只有28.1%的相似性。主要原因是AiECSOD的信号肽和肝磷脂结合区域在各物种中无同源性。其次，采用qRT-PCR（quantitative real time PCR）方法分析三种SOD基因在不同组织中的表达情况，结果表明三种SOD基因的组织表达有所差异。AiCuZnSOD基因在鳃中表达水平最高，其次是血细胞和性腺，在外套膜、闭壳肌和肝胰脏表达水平较低。AiMnSOD基因在鳃中表达水平最高，其次是外套膜，在血细胞、性腺，而在肝胰脏和闭壳肌表达较弱。AiECSOD基因在血细胞中表达水平最高，其次是肝胰脏，在鳃、闭壳肌表达水平较低，而性腺和外套膜没有检测到。同时，采用qRT-PCR对鳗弧菌Vibrio angullarum感染后海湾扇贝血细胞中三种SOD基因mRNA表达变化进行了检测。AiCuZnSOD表达量在各个时间段没有显著差异(P > 0.05)。AiMnSOD的表达量在1.5 h时略有下降，在3 h时达到最高表达量，是空白组（0h）的3倍(P < 0.01)，从6 h到24 h表达量逐渐下降，24 h时表达量是空白组的1.6倍，24 h到48 h又稍有升高。AiECSOD的表达量在1.5 h时有所下降，是空白组的0.3倍（P < 0.05)，随后逐渐升高，在12 h时达到最高表达量，是空白组（0h）的4.5倍（P < 0.01），从24 h到48 h表达量逐渐下降并恢复到空白组的水平。在对照组，各个时间点没有显著差异（P > 0.05）。在鳗弧菌感染后，海湾扇贝三种SOD的表达并不一致，且差异比较显著。AiCuZnSOD被认为是构成性表达基因，其受外界刺激的影响最小，AiMnSOD和AiECSOD受刺激后表达上调比较明显。第三，采用Genome-walking的方法得到了海湾扇贝三种SOD基因的基因组全长和近端启动子序列并对其进行了相关分析。AiCuZnSOD的基因组序列全长为4279bp，包含有4个外显子和3个内含子。AiMnSOD的基因组序列全长为10692bp，包含有4个外显子和3个内含子。AiECSOD的基因组序列全长为5276bp，包含有5个外显子和4个内含子。三种基因外显子和内含子的结合处序列遵循-AT/GT-原则。我们把海湾扇贝SOD家族的三个基因的近端启动子进行了比较分析。发现三种SOD在靠近起始密码子的位置都有Oct-1结合位点。三种SOD共有的转录位点有：Oct-1、C/EBPalp、Oct2.1、Sp-1和GATA-1。AiCuZnSOD和AiMnSOD共有的转录位点有：ICSBP、Ftz、TATA-box、C/EBPbeta和Antp。AiCuZnSOD和AiECSOD共有的转录位点有：AP-1和NFκB。AiMnSOD和AiECSOD共有的转录位点有：GR和ER。AiCuZnSOD独有的位点有：SRF、YY-1和NF-1。AiMnSOD独有的位点有：HNF-1、Hb、MEB、NF-muE1、Pit-1a和Eve。AiECSOD独有的位点有：CREB、RATA-alph、Kruppel-like和AP-3。此外，通过构建原核表达载体，本研究对AiCuZnSOD和AiECSOD基因进行了体外重组表达，并对纯化的重组蛋白进行了酶活分析。酶活分析表明，重组AiCuZnSOD蛋白有较高的酶活和稳定性。最后，我们对海湾扇贝三种SOD基因的部分区域，包括启动子、编码区，部分内含子区域进行了SNP检测，并对SOD基因部分SNP位点多态性和鳗弧菌敏感性进行了相关分析。三种SOD基因中，我们共发现了59个SNP位点，其中AiECSOD的SNP位点最多，特别是在启动子区，AiCuZnSOD和AiMnSOD多态性较低。其中AiCuZnSOD启动子区的-1739 T-C 位点的基因型和等位基因，AiECSOD启动子区的-498 A-T和-267 G-A等位基因频率，AiECSOD的第一个外显子38 Thr-Lys的多态性在敏感和抗菌群体中存在显著差异（P < 0.05）。
其他摘要	The bay scallop Argopecten irradians, which was first introduced into China from the USA in 1982, has become one of the most important bivalves cultured in China. It now dominates scallop farming in China and accounts for about 70% of the total production. However, bay scallop cultivation has suffered seriously from high mortality. Understanding the immunity of bay scallops is crucial for managing diseases and developing sustainable scallop culture. It has been proved that one important immune defense reaction of mollusca hemocytes is phagocytosis when the organism is attacked by microorganisms or viruses. During the course of phagocytosis, the host glycolytic reactions get activated which in turns increase the consumption of oxygen and induce the production of a mass of reactive oxygen species (ROS). At the same time, the host also starts other immune response to defense the infection of pathogenys. All these immune response will need more ATP to support the energy which will also result in more ROS production from the electron transport chain. Though ROS can kill foreign invaders, the mass accumulation of these reactive molecules in organisms will damage many cell components such as lipids, proteins, and nucleic acids. So the rapid elimination of these excessive ROS is essential for the proper functioning of cells and the survival of mollusca. Superoxide dismutases (SOD) family are thought to be one of the first lines of antioxidant defense and are highly efficient in protecting cells and tissues against oxidative stress by catalyzing the dismutation of superoxide radicals to form hydrogen peroxide and molecular oxygen. To begin with, three unique and highly compartmentalized bay scallop superoxide dismutases genes (CuZnSOD, MnSOD, and ECSOD) have been cloned and charactereized. The full-length CuZnSOD cDNA of bay scallop (AiCuZnSOD) was comprised of 1047bp, containing a 459bp open reading frame (ORF) which encoded 152 amino acids. Sequence comparison showed that the AiCuZnSOD shares 77.5% and 75% similarity with CuZnSOD of Chlamys farreri and Crassostrea gigas respectively. 74.5% similarity with that of human. The full-length cDNA of MnSOD (AiMnSOD) consisted of 1207 bp with a 681 bp ORF encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the AiMnSOD shares 85% and 78.4% similarity with MnSOD of Mizuhopecten yessoensis and Haliotis discus discus, respectively. The full-length cDNA of ECSOD (AiECSOD) was 893 bp with a 657 bp ORF encoding 218 amino acids. The deduced amino acid sequence contained a putative signal peptide of 20 amino acids. Sequence comparison showed that AiECSOD had low degree of homology to ECSODs of other organisms, which shares 27.9% similarity with that of Brugia pahangi, 31.4% with similarity with Danio rerio, 28.6% with human, and only 28.1% with mollusca C. gigas. Then, the genomic length of the AiCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region contained many putative transcription factor binding sites such as ICSBP, Oct-1, Sp1, TBP, C/EBPbeta, C/EBPalp, NF-1, NFκB, GATA-1, AP-1, YY1 and SRF binding sits. The genomic length of the AiMnSOD gene was about 10692 bp containing 4 exons and 3 introns. The promoter region contained many putative transcription factor binding sites such as ICSBP, Oct-1, Sp1, TBP, C/EBPbeta, C/EBPalp, NF-muE1, GATA-1, Pit-1a, HNF-1, GLO, HOXA4 and Antp binding sits. The genomic length of the AiECSOD gene was about 5276 bp containing 5 exons and 4 introns. The promoter region contained many putative transcription factor binding sites such as c-Myb, Oct-1, Sp1, Kruppel-like, c-ETS, NFκB, GATA-1, AP-1, and Ubx binding sites. Moreover, a quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of SODs in different tissues and the temporal expression of SODs in scallop challenged with bacteria Vibrio angullarum. Higher-level mRNA expression of AiCuZnSOD was detected in the tissues of gill filaments, haemocytes and gonad. The expression of AiCuZnSOD was no significant change from 0h to 48h after the bay scallop injected with V. angullarum. Higher-level mRNA expression of MnSOD was detected in gill and mantle. The expression of AiMnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. High levels of expression were detected in haemocytes, but not in gonad and mantle. The expression of AiECSOD reached the highest level at 12 h post-injection with V. anguillarum and then returned to normal between 24 h to 48 h post-injection. The results indicated that bay scallop SODs was a constitutive and inducible protein. It could play an important role in the immune responses against V. anguillarum infection in different way. In addition, expression, purification of recombinant and the enzyme activity assay confirm AiCuZnSOD and can express in E. coli with high enzyme activity and the enzyme is stable in vitro. AiECSOD can also expresss in E.coli but with low enzyme activity. Finally, three SOD isoforms polymorphism and the possible association of SNPs with resistance/susceptibility to bacteria Vibrio angullarum infection were also studied. In promoters, exons and partially introns of three SODs, 59 SNPs were discovered. Compare to AiCuZnSOD and AiMnSOD, AiECSOD has the most SNPs, especially in promoter. Among them, -1739 T-C SNP in AiCuZnSOD promter, -498 A-T and -267 G-A SNPs in AiECSOD promoter were significant associations with resistance/susceptibility to bacteria V. angullarum infection (P<0.05). The polymorphism of 38 Thr-Lys in the extracellular targeting sequence in AiECSOD is also associated.
页数	149
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1359
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	包永波. 海湾扇贝超氧化物歧化酶家族基因结构、表达和多态性分析[D]. 海洋研究所. 中国科学院海洋研究所,2009.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
10001_20061800681205（3483KB）			限制开放	--	浏览