【作者】 任洪林；

【导师】 王克坚；

【作者基本信息】 厦门大学 ， 环境科学， 2008， 博士

【摘要】 鲍以其丰富的营养价值在民间一直被认为是与鱼翅、燕窝齐名的营养食品。养殖鲍市场价格高且稳定,销路畅通,利润空间大,深受水产养殖户的欢迎,是我国重要的海水养殖经济物种。杂色鲍(Haliotis diversicolor Reeve,1846)隶属于软体动物门,腹足纲,前鳃亚纲,原始腹足目,鲍科,是我国南方鲍种,因其生长迅速,一直在南方沿海海域得到广泛的养殖。但近年来由于养殖海域水质影响及养殖鲍种自身退化,使得人工养殖杂色鲍抗病力低,时有疫病发生,给养鲍业带来很大的经济损失。因此,寻求免疫防治是保障鲍养殖业健康发展的重要措施,但迄今有关鲍免疫机制的研究报道较少。本项研究应用抑制性差减杂交(SSH)技术筛选细菌攻毒杂色鲍免疫相关基因,为深入探讨鲍抗细菌感染免疫机制奠定基础。1成功构建细菌攻毒杂色鲍血淋巴细胞SSH cDNA文库血淋巴细胞是鲍免疫系统中重要的免疫细胞,本研究以雌性杂色鲍为对象,以副溶血弧菌(Vibrioparahaemeolyticus)、大肠杆菌(Escberichia coli)、溶壁微球菌(Micrococcuslysodeikticus)、金黄色葡萄球菌(Staphylococcus aureus)、表皮葡萄球菌(Staphylococcus epidermidis)五种细菌混悬液为攻毒菌,利用SSH技术,成功构建细菌攻毒杂色鲍血淋巴细胞SSH cDNA文库,该文库容量为1.37×10~6 cfu,重组效率为98.18%。2克隆获得111个杂色鲍血淋巴细胞表达基因随机测序435个重组子,经序列分析和拼接,总计克隆获得111个杂色鲍血淋巴细胞表达基因。利用AmiGO基因本体释义工具,将其分为11个基因群,11个基因(9.9%)参与细胞蛋白质代谢过程;8个基因(7.2%)参与产生前体代谢物和能量的生物学过程;5个基因(4.5%)参与异生质代谢等其他细胞代谢过程;10个基因(9.0%)参与细胞组分生物合成与装配过程;6个基因(5.4%)参与机体信号传导;8个基因(7.2%)参与生物调控;8个基因(7.2%)参与免疫系统过程;8个基因(7.2%)参与应激反应过程;4个基因(3.6%)分别参与不同的生物学过程被合为一个其他功能基因群;40个基因(36.0%)属于完全未知功能的基因;3个基因(2.7%)是rRNA基因。BLAST同源序列搜索分析发现25个基因(22.52%)在GenBank数据库中搜索到来源于腹足纲动物的同源基因信息,其他86个基因(77.48%)均是首次在腹足纲动物中发现。3确定了52个上调表达的基因利用半定量PCR和real-time PCR两种基因差异表达分析方法共同证实52个基因在细菌攻毒36-40 h的杂色鲍血淋巴细胞中表现不同程度的差异表达。其中47个基因(42.34%)在细菌攻毒血淋巴细胞中上调表达,5个基因(4.5%)在生理盐水对照组上调表达。4扩增获得86个基因的特异基因组DNA片段通过PCR扩增基因组DNA片段和BLASTn同源搜索证实所克隆的杂色鲍血淋巴细胞表达基因无细菌基因组DNA污染。86个目的基因可扩增出特异基因组DNA片段,其中46个基因的基因组DNA在扩增引物之间无内含子序列;40个基因的基因组DNA序列在扩增引物之间存在内含子。通过Southern blotting和DNA测序证实β-thymosin基因(Thym-β)的基因组DNA序列至少含有两个基因组拷贝,其中一个基因拷贝不含内含子,另一个基因拷贝含有两个内含子,第一内含子477 bp,第二内含子2818bp。5揭示了16个基因的全长cDNA序列利用SMART技术和游离PCR、锚定PCR原理,较为经济的批量扩增克隆了16个杂色鲍血淋巴细胞表达基因全长cDNA序列,其中GlSTrs、Profilin、TIMp、PreP2、Thym-β、Ferritin、Calp、Hypo等8个基因是在细菌攻毒36-40 h杂色鲍血淋巴细胞中上调表达基因。6分析了14个基因在鲍体内的差异表达特性利用半定量PCR和荧光定量PCR方法分析GlSTrs、Thym-β、AlInFa、CYP7A1、KLF、ferritin、Hypo2、MMPvar、CDD、SOCS-2、Profilin、Hypo、TIMp、UbCoE等14个基因的差异表达特性。证实GlSTrs、CYP7A1、Hypo2、TIMp、CDD、A1InFa、MMPvar、Hypo等8个基因受细菌诱导时间影响在血淋巴细胞内显著的上调表达。本项研究首次从基因组学的角度出发,分析杂色鲍响应细菌攻毒血淋巴细胞差异表达基因。鲍免疫相关基因的研究为进一步探讨鲍抗感染免疫机制和提高抗病力奠定分子生物学基础,也为深入理解低等动物免疫机制提供新的研究切入点。更多还原

【Abstract】 Abalones are one of the largest marine gastropod mollusks and they are a economically important seafood in aquaculture worldwide.However, bacterial epidemic infection has been reported in China and other countries in recent years and mass mortality in abalones causes significant economic losses.For this reason,a large scale screening of immune-related abalone genes is necessary.A better understanding of the immune responses in variously colored abalone(Haliotis diversicolor Reeve,1846) after bacterial infection is important to protect this animal from diseases in the farm.In order to find differentially expressed genes in bacteria-challenged abalones,a forward subtractive suppression hybridization(SSH) library with content of 1.37×10~6 cfu was constructed from haemocytes of H. diversicolor challenged with five bacterial species including two Gram negative bacteria(Escherichia coli and Vibrio parahaemeolyticus) and three Gram positive bacteria(Staphylococcus aureus,Micrococcus lysodeikticus and Staphylococcus epidermidis),and the potential ESTs or gene sequences were randomly screened on the middle sequencing scale(435 clones).A total of 435 clones in the SSH library were sequenced and 111 genes were recognized based on BLAST searches in NCBI and were categorized in association with different biological processes using AmiGO against the Gene Ontology database.Among seventy-one genes(64.0%) involved in different biological processes,24 genes(21.6%) were categorized as a group in association with cellular metabolic processes,of which 11 may involve in a cellular protein metabolic process(9.9%),8 were associated with a generation of precursor metabolites and energy(7.2%) and 5 with other cellular metabolic processes(4.5%);10 were related to cellular component organization and biogenesis(9.0%);4 were associated with other functions(3.6%);6 were listed as a signal transduction group (5.4%);8 were involved in a biological regulation group(7.2%),and the equal number of genes in immune system processes(7.2%) and response to stimuli(7.2%);3 were rRNA genes.In addition,40 genes(36.0%) were classified as an unknown-function gene group.Only 25 of the 111 genes (22.52%) had previously been reported in other gastropods,and most of the screened genes showed less similarity to known sequences based on BLASTn results,suggesting that at least 86 genes were found for the first time in H.diversicolor.Both semi-quantitative PCR and quantitative real-time PCR were used to validate the differential display expressions of genes which were screened from the SSH library.Fifty-two genes among 111(46.85%) were confirmed to be differentially expressed in the library,and 47 out of 111(42.34%) were up-regulated after bacterial challenge,while only five out of 111(4.5%) were up-regulated in the unchallenged controls.Among the 111 genes,55(49.55%) could not be confirmed to show differential expression patterns in either the bacteria-challenged or the unchallenged groups,and four(3.60%),often regarded as internal standard housekeeping genes,were obtained.In particular,30 genes(GlSTrs,CYP7A, Endoph,SOCS-2,Hypo2,PreP2;TroponinT,CDD,MEP and Unk29,AlInFa, MyosinLC,MMP1,MMPvar,Calp,Astacin-like,Hypo,Unk,Unk4,Unk5,Unk19, Unk24,Unk21,Unk28,Unk31,Unk32,Unk35,Unk39,HDr4CJ516, HDr2CJ15),encoding proteins involved in cellular metabolic processes; cellular component organization and biogenesis;signal transduction and biological regulation;immune defense and response to stimuli;other functions and unknown functions,were confirmed to be distinctly up-regulated in the bacteria-challenged group by both assays,and 17 genes (UbCoE,RPN2,ATPsynSub9,Profilin,TIMp,KLF,Thym-β,Caspase8, Ferritin,Tis11FPL,GSTisof,Hsp40,MnSoD,Unk2,Unk3,Unk17 and Unk34) were to some extent up-regulated in the bacteria-challenged group.In addition,five genes(FASCIN,AlDe,CuZnD,Unk11 and Unk7) were up-regulated in the unchallenged group.To exclude the possibility of contamination in abalone cDNAs from SSH by bacterial sequences,we designed 101 pair primers specific to the translatable genes indicated by SSH to amplify the genomic DNA of H. diversicolor.Specific genomic DNA fragments were obtained,especially for the genes showing no significant similarity.Cloned genomic DNA ofβ-thymosin suggested that two copies localed on chromosomal DNA.One copy is without intron,and the other is with two introns(477 bp and 2818 bp).Sixteen complete cDNAs of the genes screened from the SSH library were elucidated,including 8 genes of GlSTrs,Profilin,TIMp,PreP2,Thym-β, Ferritin,Calp and Hypo up-regulated in haemocytes of bacteria-challenged abalones during 36 h to 40 h.The differential time-respones relationship of 14 genes expression induced by bacteria in haemocytes of H. diversicolor were studied and the results of 8 genes including GlSTrs, CYP7A1,Hypo2,TIMp,CDD,AlInFa,MMPvar,Hypo showed to be distinctly up-regulated in haemocytes corresponding to time of bacterial challenge.In summary,this study represents the first large-scale investigation of immune-related genes in bacteria-challenged abalones.By SSH, differential expression patterns of genes were screened specifically related to bacterial stimulation and these genes were then categorized into 11 distinct functional groups.Eighty-six genes were for the first identified among 111 screened in H.diversicolor,of which 52 genes were confirmed to show the differential expression patterns using both semi-quantitative PCR and quantitative real-time PCR.A total of 30 genes were validated to be distinctly up-regulated in bacteria-challenged abalones,encoding proteins involved in cellular metabolic processes; cellular component organization and biogenesis;signal transduction and biological regulation;immune defense and response to stimuli;other functions and unknown functions.To our knowledge,this is the first report to unveil multiple up-regulated genes with differential expression patterns involving various biological processes in bacteria-challenged H.diversicolor.It is anticipated that these results will be useful in elucidating the immune mechanism in bacteria-challenged H.diversicolor. However,because it is not clear that the genes screened in this study take part in immune processes,further research may focus on revealing the biological functions of genes screened in the SSH that are up-regulated.We anticipate that this work will be helpful in facilitating future study of the immune response of H.diversicolor,and acquiring new insights into the mechanism involved.更多还原