低渗诱导凡纳滨对虾肝胰腺消减文库的构建及其表达序列标签分析

Construction of the Hepatopancreas Subtractive cDNA Library of Litopenaeus vannamei Induced by Hyposmotic Stress and ESTs Analysis

初始体重（0．27±0．01）g的凡纳滨对虾（Litopenaeus vannamei）分别于盐度2和30中饲养56d，采用抑制性消减杂交技术构建低渗胁迫诱导的凡纳滨对虾肝胰脏差异表达基因消减cDNA文库。消减杂交效率分析显示，正反向消减文库差异表达的基因各富集大约25和2^5-10倍。从正反向两个文库中随机挑选200个阳性克隆进行测序（正向文库80个，反向文库120个），共获得179条序列长度大于100bp的ESTs序列，经CAP3在线拼接程序得到73条Unigenes（19条contigs和54条singlets）。生物学信息分析表明，这些Unigenes所代表的相应基因的功能包括免疫相关蛋白、代谢相关蛋白、核蛋白、细胞凋亡和转移蛋白。这些基因信息表明，凡纳滨对虾在低盐胁迫诱导所导致的生理变化非常复杂，构建的差异表达cDNA文库可较好地反映低盐胁迫对凡纳滨对虾影响的基因信息。

To investigate the effect of low salinity stress on gene expression of hepatopancreas in shrimp, we applied suppression subtractive hybridization （SSH） in juvenile white shrimp （Litopenaeus vannamei） exposed to low salinity. The shrimps （initial body weight, 0.27 _ 0.01 g） cultured at salinity 2 and salinity 30 for 56 days respectively, forward and reverse subtractive cDNA libraries of hepatopancreas in shrimp cultured at these two salinities were constructed. Subtraction efficiencies were about 2^5 and 2^5-10-fold enrichment in hepatopancreas of forward and reverse cDNA libraries. A total of 200 （80 from forward, 120 from reverse） randomly selected clones was sequenced and acquired 179 ESTs with more than 100 nucleotides. 19 contigs and 54 singletons were generated from a total of 73 Unigenes after CAP3 online sequence assembly program. Results of bioinformatics analysis indicated that the unigenes encoded proteins corresponded to a wide range of functions including immune-related proteins and enzymes, metabolism-related enzymes, ribosomal proteins, apoptosis-related proteins and transfer proteins, which illuminated that the corresponding physiology response induced by low salinity was very complicated.The cDNA subtractive library could well reflect the gene information of L. vannamei induced by long-term low salinity-stress, results of which provide the basic data to research of relationship between environmental stress and gene expression of liver.