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本研究以凡纳滨对虾(Litopenaeus vannamei)为对象,分别以低盐度胁迫下(4 ppt)凡纳滨对虾鳃丝cDNA为检测子,正常海水中凡纳滨对虾鳃丝cDNA为驱动予,采用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建了急性低盐胁迫诱导下凡纳滨对虾鳃丝消减cDNA文库。文库的重组率均高于95%,插入片段集中在250~750 bp之间,随机测序后在线拼接得到15组片段重叠群和37个单一序列共52个非重复序列。同源检索发现30个非重复序列与GenBank中的已知基因序列有较高的相似性。按差异表达基因编码的蛋白具体功能可分为6小类,依次为:(1)离子通道及转运蛋白;(2)蛋白合成翻译及转录因子;(3)应激抗氧化因子;(4)能量代谢;(5)信号受体和转导;(6)细胞纤维及骨架蛋白,所占比例分别为23.3%、20.0%、20.0%、16.7%、10.0%和10.0%。经功能聚类分析发现文库中含有大量离子通道蛋白及功能转运酶、胞内信号传导分子、细胞骨架蛋白等渗透调节和应激适应性相关基因,表明急性低盐胁迫诱导凡纳滨鳃丝抑制性消减杂交cDNA文库构建成功。
In this study, Litopenaeus vannamei was used as a probe to detect the gill cDNA of Litopenaeus vannamei under low salinity stress (4 ppt) Suppression subtractive hybridization (SSH) technique was used to construct the gill-subtracted cDNA library of Litopenaeus vannamei induced by acute low salt stress. The recombination rate of the library was higher than 95%. The inserted fragments were concentrated in the range of 250-750 bp. After random sequencing, 15 fragments of contigs and 37 non-repetitive sequences of 37 single sequences were obtained. Homology search found 30 non-repetitive sequences with the known gene sequences in GenBank have a higher similarity. The specific functions of proteins encoded by differentially expressed genes can be divided into six subgroups, which are: (1) ion channels and transporters; (2) protein synthesis translation and transcription factors; (3) stress and antioxidant factors; (4) Energy metabolism; (5) signal transducers and transducers; (6) cellular fibers and skeletal proteins, accounting for 23.3%, 20.0%, 20.0%, 16.7%, 10.0% and 10.0% respectively. The functional cluster analysis showed that the library contains a large number of ion channel proteins and functional transporter enzymes, intracellular signal transduction molecules, cytoskeletal proteins such as osmoregulation and stress adaptation related genes, indicating that acute low salt stress induced glossophane gill inhibition Sex subtractive hybridization cDNA library was constructed successfully.