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为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期c DNA为检测子(tester)、双核菌丝期c DNA为驱赶子(driver),采用抑制性消减杂交法(suppression subtractive hybridization,SSH)构建了糙皮侧耳SSH c DNA文库。菌液PCR验证SSH c DNA文库插入c DNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列(expressed sequence tag,EST),重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH c DNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。
In order to analyze the genes differentially expressed in the basal stage and the mycelial stage of Pleurotus ostreatus, we used c-DNA as tester and mitochondria c DNA as driver, using suppression subtractive hybridization suppression subtractive hybridization (SSH)). Bacterial PCR was used to verify the SSH c DNA library inserted into the c DNA fragment, picked 2 055 differential transformants, the differential transformants by 3 reverse Northern hybridization screening, access to 423 significant differences in signal cloning; positive cloning sequencing, A total of 206 expressed sequence tags (ESTs) were obtained from the NCBI database Blastn and Blastx. After repeated sequence deletion, 46 genes were involved in the cell rescue and defense, energy metabolism, transcription and protein regulation, membrane proteins And signal transduction, 18 genes encode unknown function putative proteins and 5 new genes without any homology. Ten differential expression genes were selected for semi-quantitative RT-PCR and the expression level of these sequences at the primordial stage was significantly higher than the mycelial stage. The results showed that the SSH-DNA library of Pleurotus ostreatus and mycelial stage was successfully constructed in this study, which laid the foundation for the further isolation of the genes related to growth and development of Pleurotus ostreatus and the study on the mechanism of development of Pleurotus ostreatus.