应用cDNA微阵列芯片技术筛选猪蛔虫性别差异表达基因的研究

Profiling and Identification of Gender-specific Expressed Genes in Ascaris suum by cDNA Microarray Analysis

采用cDNA微阵列芯片技术,从所构建的猪蛔虫雌、雄成虫cDNA消减文库分别挑取1 044和1 119个克隆,PCR扩增其插入片段,经纯化后点样于预先处理好的基片上(双点杂交),制备成cDNA微阵列芯片。将分别标记荧光素Cy3-dUTP和Cy5-dUTP的雌虫和雄虫cDNA探针,与制备好的cDNA芯片杂交(平行进行反标杂交试验)。根据每个点杂交后的Ratio值,筛选出双点杂交和正反标中都同时具有表达差异的基因克隆共1 559个。将表达差异最明显的前831个克隆进行测序,获得720个有效序列,经生物信息学分析发现,雄虫特异表达的主要精子蛋白和雌虫特异表达的卵巢信息蛋白的基因序列多数与新杆属线虫存在同源性,有31个可能是新的ESTs。性别差异表达基因及其相关生物信息的获得为下一步研究基因功能奠定了基础。

Gender-specific gene expression in Ascaris suum was investigated by eDNA mieroarray analysis. A total of 2 163 clones （1 044 female and 1 119 male） were selected from the male and female A. suum cDNA libraries constructed using the technique of suppression subtractive hybrid ization （SSH）. Insert fragments of them were amplified by PCR, the PCR products were then purified and robotically printed onto aminosilane-coated glass slides, and each clone was spotted twice in the same slide. The eDNA arrays were hybridized with Cy3-dUTP and Cy5-dUTP fluorescent-labeled probes from female and male cDNA, respectively. A ＂dye swap＂ experiment was performed simultaneously. The hybridization signal of each spot was measured, normalized, and expressed as a Ratio. In total, 1 559 clones were identified which showed differential expression for each of the four signal values （for the two repeats and the two dye swaps） according to their Ratios （Ratio=CyS/Cy3^＊ ,less 0.5 or more 2）. Expression profiling revealed that 831 of these clones were considered to be significantly differentially expressed, and then were sequenced. A total of 720 valid ESTs were obtained and 31 of which were considered to represent new genes. ESTs representing genes encoding male-specific major sperm proteins （MSPs） and female-specific ovarian message proteins were abundant and showed significant homology to those of Caenorhabditis. Identification of gender-specific genes provides foundation for further studies on the potential functions of key gender-specific genes.