低温处理与常温凡纳滨对虾基因差异表达文库的构建及分析

目的]筛选出对虾耐寒相关基因,为今后进一步研究对虾耐寒分子机理提供科学依据.方法]利用SSH技术构建低温处理与常温凡纳滨对虾的基因差异表达文库,并通过斑点杂交、克隆测序分析及候选基因RT-PCR扩增等对差减获得的耐寒相关功能基因进行验证.结果]在构建的正、负文库768个克隆中有152个为低温高表达克隆、331个为常温高表达克隆;将正文库100个克隆拼接可获得ESTs同源群39个,其中已知基因29个,主要涉及能量代谢、表达调控、信号传导、细胞免疫、抗细胞凋零、蛋白质加工及其他基因等;将负文库50个克隆拼接可获得ESTs同源群18个,其中已知基因17个,主要涉及细胞免疫、维持细胞代谢、物质转运、信号传导及其他基因等.经RT-PCR验证,从正文库中筛选出的HSPB1、NGT4和NGT7基因在低温条件下的表达量显著高于常温.结论]综合运用SSH、斑点杂交、克隆测序和RT-PCR可有效筛选并验证凡纳滨对虾耐寒相关基因,为揭示对虾耐寒分子调控机制奠定基础.

Construction and analysis of SSH library of pacific white leg shrimp Litopenaeus vannamei exposed to low temperature

Objective ]Some cold related genes of Litopenaeus vannamei were screened to provide scientific refer- ences for further study on cold resistant molecular mechanism of Litopenaeus vannamei. Method]Two gene differential expression libraries for the low-temperature processing and the room-temperature were constructed by Suppression Sub- tractive Hybridization （SSH）, furthermore, some cold-related genes by dot blot hybridization were achieved and verified by ESTs sequence and analysis, Real-time PCR and so forth. Result ]768 clones were picked from the forward and reverse library randomly and verified by dot blot hybridization, 152 of which were high expression clones at the low- temperature group and 331 of which were high expression clones at the ruom-temperature group. 39 Contigs were ob- tained after splicing 100 ESTs sequenced from the forward library, including 29 known genes, which mainly related to energy metabolism, expression and regulation, signal transduetion, cellular immunity, anti apoptosis, protein process- ing and other. 18 Contigs were obtained after splicing 50 ESTs sequenced from the reverse library, including 17 known genes mainly related to cellular immunity, the maintenance of cell metabolism, material transport, signal transducfion and others. It was confirmed from RT-PCR that the expression quantities of the HSPB1, NGT4 and NGT7 genes select- ed from forward library were much larger at the low temperature than they were at the room-temperature. Conclusion ] Through the comprehensive utilization of SSH, dot blot hybridization, ESTs sequence and analysis, RT-PCR and so forth, effective screening and verification of Litopenaeus vannamei cold-related genes can be achieved to provide basic materials for further study on gene function and cold resistant molecular mechanism of Litopenaeus vannamei.