三角帆蚌肝脏消减cDNA文库的构建与分析
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摘要
三角帆蚌(Hyriopsis cumingii)是中国特有的最主要的淡水育珠蚌种。三角帆蚌瘟病是严重危害淡水珍珠生产的传染性蚌病,该病传染快、死亡率高、治疗难度大。20世纪80年代开始的三角帆蚌瘟病病原相关研究一直存在病毒与细菌两类病原的报道。本研究以2005年9月采集的三角帆蚌瘟病典型症状病蚌为基本材料,为探索三角帆蚌瘟病的抗病育种技术途径与方法,应用抑制性消减杂交技术(SuppressionSubtractive Hybridization,SSH),建立了三角帆蚌肝脏cDNA文库,筛选分析三角帆蚌瘟病的免疫相关因子。
1.三角帆蚌肝脏消减cDNA文库构建
采用抑制性消减杂交技术,构建了健康三角帆蚌和抗病三角帆蚌(攻毒后健康存活者)肝脏的消减cDNA文库,随机挑取300个cDNA克隆进行了单向测序,共获得有效cDNA序列214个。
2.三角帆蚌肝脏消减cDNA文库序列分析
序列同源性搜索结果显示,在214个有效cDNA序列中,有120个克隆和GenBank中的已知功能的序列具有较高的同原性(E值＜-6);30个则与未知功能的开放阅读框具有较高的相似性,称之为假定蛋白;21个序列则没有任何相似的序列,很有可能是以前没有鉴定过的EST序列或是5'-或3'-UTR序列。214个已知功能的EST分别属于98个不同的基因,均为三角帆蚌的首次报道。
根据Adams对功能基因的分类方法,所获得EST分为8类:细胞分裂相关、细胞结构与运动相关、代谢相关、信号传导相关、细胞防疫相关、基因与蛋白表达相关、其它功能蛋白和未知功能蛋白等。其中细胞防御等免疫相关因子序列的克隆数达40个,占总克隆数的18.2%。
Hyriopsis cumingii is one of the most important endemic freshwater pearl species in China. H. cumingii Plague is an infectious disease and spreads fast. It severely affects the production of freshwater pearl mussels by causing high mortality with no affective treatments. Studies on H.cumingii plague pathogen, including pathogenic bacteriology and virology, has reported since 1980s. In this study, mussel samples with typical symptoms of H. cumingii plague were collected in September 2005. To establish technology and methodology of genetic breeding resistant to H.cumingii plagues, the newly developed technique of suppression subtractive hybridization (SSH) was used to construct a cDNA library of the H.cumingii liver and screened for immune related factors associated with H.cumingii plagues.
1. Construction of subtracted liver cDNA library in Hyriopsis cumingii
Subtracted liver cDNA library of healthy and disease-resistant H.cumingii (survivorsafter infected) were constructed using the suppression subtractive hybridization technology (SSH). 214 out of the 300 positive cDNA clones randomly selected for sequencing were effective in obtaining of cDNA sequences.
2. Sequencing analysis of positive clones in Hyriopsis cumingii
Sequence homology search showed that 120 out of the 214 effective cDNA sequences were homologous with high sequence identities to known sequences of functional proteins in the GenBank (E value≥6). Sequences of 30 positive clones had high similarities with open reading frame (ORF) of molecules of unknown function referred as hypothetical protein in the database. 21 sequence showed no significant similarity to any other sequences, which might be a novel EST sequence and a potential 5'- or 3'- UTR sequence in the genome that had been reported previously. 21 known function EST for 40 different genes were reported in H.cumingii.for the first time in this study.
According to the classification of functional genes by Adam, the identified EST in H.cumingii fell into eight categories relevant to cell division, cellular structure and movement, metabolism, signal transduction, cell immune-action, gene and protein expression associates and other proteins with unknown functions. 40sequences, 18.2% of the total analyzed clones, reported in this study are related to the immune factors of cell recovery.
1.三角帆蚌肝脏消减cDNA文库构建
采用抑制性消减杂交技术,构建了健康三角帆蚌和抗病三角帆蚌(攻毒后健康存活者)肝脏的消减cDNA文库,随机挑取300个cDNA克隆进行了单向测序,共获得有效cDNA序列214个。
2.三角帆蚌肝脏消减cDNA文库序列分析
序列同源性搜索结果显示,在214个有效cDNA序列中,有120个克隆和GenBank中的已知功能的序列具有较高的同原性(E值＜-6);30个则与未知功能的开放阅读框具有较高的相似性,称之为假定蛋白;21个序列则没有任何相似的序列,很有可能是以前没有鉴定过的EST序列或是5'-或3'-UTR序列。214个已知功能的EST分别属于98个不同的基因,均为三角帆蚌的首次报道。
根据Adams对功能基因的分类方法,所获得EST分为8类:细胞分裂相关、细胞结构与运动相关、代谢相关、信号传导相关、细胞防疫相关、基因与蛋白表达相关、其它功能蛋白和未知功能蛋白等。其中细胞防御等免疫相关因子序列的克隆数达40个,占总克隆数的18.2%。
Hyriopsis cumingii is one of the most important endemic freshwater pearl species in China. H. cumingii Plague is an infectious disease and spreads fast. It severely affects the production of freshwater pearl mussels by causing high mortality with no affective treatments. Studies on H.cumingii plague pathogen, including pathogenic bacteriology and virology, has reported since 1980s. In this study, mussel samples with typical symptoms of H. cumingii plague were collected in September 2005. To establish technology and methodology of genetic breeding resistant to H.cumingii plagues, the newly developed technique of suppression subtractive hybridization (SSH) was used to construct a cDNA library of the H.cumingii liver and screened for immune related factors associated with H.cumingii plagues.
1. Construction of subtracted liver cDNA library in Hyriopsis cumingii
Subtracted liver cDNA library of healthy and disease-resistant H.cumingii (survivorsafter infected) were constructed using the suppression subtractive hybridization technology (SSH). 214 out of the 300 positive cDNA clones randomly selected for sequencing were effective in obtaining of cDNA sequences.
2. Sequencing analysis of positive clones in Hyriopsis cumingii
Sequence homology search showed that 120 out of the 214 effective cDNA sequences were homologous with high sequence identities to known sequences of functional proteins in the GenBank (E value≥6). Sequences of 30 positive clones had high similarities with open reading frame (ORF) of molecules of unknown function referred as hypothetical protein in the database. 21 sequence showed no significant similarity to any other sequences, which might be a novel EST sequence and a potential 5'- or 3'- UTR sequence in the genome that had been reported previously. 21 known function EST for 40 different genes were reported in H.cumingii.for the first time in this study.
According to the classification of functional genes by Adam, the identified EST in H.cumingii fell into eight categories relevant to cell division, cellular structure and movement, metabolism, signal transduction, cell immune-action, gene and protein expression associates and other proteins with unknown functions. 40sequences, 18.2% of the total analyzed clones, reported in this study are related to the immune factors of cell recovery.
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