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,金属结合蛋白,肌动蛋白等。以上研究结果为筛选厚壳贻贝免疫相关的新基因,深入研究厚壳贻贝免疫相关因子的分子多样性及免疫机制奠定了基础。
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.
出处
《海洋与湖沼》
CAS
CSCD
北大核心
2009年第5期603-607,共5页
Oceanologia Et Limnologia Sinica
基金
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
