| 摘要: |
| 采用TRIZOLReagent 提取厚壳贻贝血细胞总RNA, 分离纯化mRNA, 在此基础上以pCMV sport6 质粒为载体通过Gateway 技术构建了高质量的厚壳贻贝血细胞cDNA 文库, 以期获得厚壳贻贝血细胞的EST 序列标签以及可能的免疫相关功能基因序列。从文库中随机挑选28 个克隆进行序列测定及在线BLAST 分析。结果表明, 在28 个随机克隆中, 有4 个克隆为rRNA 基因, 7 个克隆为未知基因, 推测是新基因, 17 个克隆为功能基因, 包括脱氢酶, 细胞色素氧化酶, 热休克蛋白
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| 关键词: 厚壳贻贝, 血细胞, 免疫因子, cDNA 文库, EST |
| DOI：10.11693/hyhz200905013013 |
| 分类号: |
| 基金项目:浙江省科技厅面上科研农业项目, 2008C22026 号; 浙江省教育厅重点项目, 20070430 号; 浙江省科技厅新苗人才计划项目, 2008R40G2110003 号。 |
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| CONSTRUCTION OF HAEMOLYMPHE cDNA LIBRARY OF MYTILUS CORUSCUS AND SCREENING OF EXPRESSED SEQUENCE TAGS |
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WANG Ri-Xin1, LIAO Zhi2, LIU Mei2, LU Tao2, WU Mei2, HE Guang-Yuan3
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1.China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology (HUST), Laboratory of Marine Biology Resources and Molecular Engineering, Ocean Science College, Zh;2.Laboratory of Marine Biology Resources and Molecular Engineering, Ocean Science College, Zhejiang Ocean University;3.China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology (HUST)
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| Abstract: |
| As one of major artificial breeding marine mollusks in South China, Mytilus coruscus is an economically important species, and its antimicrobial defense has been an attractive issue to researchers. The Immunity of M. coruscus is related to various immune molecules in plasma, of which antimicrobial peptide is a strong defensin against invasive or non-invasive bacteria, and is intact to eukaryotic cells, which makes the peptide a candidate molecular for developing new biological antibiotics. To obtain the expressed sequence tags (ESTs) and full-length sequences of potential functional-genes, the total RNA of M. coruscus hemocytes was extracted with Trizol reagent, and purified in chromatography with oigo (dT) cellulose. In gateway technology, a high-quality cDNA library of M. coruscus haemolymphe was constructed with pCMV sport6 as the vector. A number of clones were randomly-selected, sequenced, and online-analyzed with BLAST. The result show that among the 28 random clones, four were characterized as rRNA genes, seven were unknown genes (presumably novel ones), and 17 were functional genes (coded for proteins including dehydrogenases, cytochrome oxidases, heat shock proteins, metal-binding proteins and actins etc.); some of them are immunity-related. The construction of high-quality cDNA library of the haemolymphe provided a tool to screen out more immunity-related functional genes and to probe molecular diversity of immunity-related factors and the immunologic mechanism.
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| Key words: Mytilus coruscus, Haemolymphe, Immune factor, cDNA library, EST |
