本研究以几纳滨对虾(Lito penaeusvunnmei)为对象.分别以低盐度胁迫下(4ppt)凡纳滨对虾鳃丝cDNA为检测予,正常海水中凡纳滨对虾鳃丝cDNA为驱动子,采用抑制性消减杂交(suppressionsubtractive 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250~750bp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j2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舍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几纳滨鳃丝抑制性消减杂交cDNA文库构建成功。
A suppression subtractive hybridization (SSH) cDNA library of postgills in Litopenaeus van- ,amei induced by acute low salinity stress was constructed in this study. The postgills of L. vannamei in hypo-osmotic stress (4 ppt) were used as the monitoring and those in normal seawater (31 ppt) as the driver element, cDNA clones were selected from library and then sequenced. These sequences were analyzed using BLASTX software and classed for the results. The recombinant efficiency of cDNA li- brary was higher than 95o/00 and the inserted fragment was concentrated between 250~750 bp. Total 81 expressed sequence tags (ESTs) were obtained by randomly sequencing, and 52 unigenes including 15 contigs and 37 singletons after CP3 online matching were gained by clustering and assembling. The re sults of sequence homology search showed that there were 80 known with significant homology to Gen- bank database sequences that were identified, while 22 unigenes did not match any sequences in the datebase and presumably represented unidentified cDNAs. The clusters were concluded into 6 catego- ries according to their translated protein functions. They were ion channels and transport proteins (1) protein synthesis, translation and transcription factor (2) stress resistance and antioxidant factors (3) energy metabolism (4) signal receptor and transduction (5) cell fibrin and cytoskeletal proteins (6) which are account for 23.3%, 20.0%, 20.0%, 16.7%, 10.0%, and 10.0% respectively. A lot of os- moregulation ralated genes were discovered and identified and the SSH cDNA library of postgills in Litoperzaeus vannamei induced by acute low salinity stress were constructed successfully.