糙皮侧耳原基期差异表达基因分析

Differential gene expression analysis of the primordium stage of Pleurotus ostreatus

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 isolate the differentially expressed genes in the primordium stage of Pleurotus ostreatus, the suppression subtractive hybridization （SSH） cDNA library was constructed using the cDNA from dikaryotic mycelium stage as a driver and the cDNA from primordium stage as a tester. In total, 2 055 positive clones were selected after the inserted fragments of the library were confirmed by bacterial colony PCR. There were 423 significantly differently expressed clones among 2 055 positive clones after three times of reverse Northern blot differential screening. The sequences were analyzed through Blastn and Blastx against the NCBI database after the positive clones being sequenced, and then 206 differentially expressed sequences were obtained.After the repeated sequences being removed, 46 genes were identified which were putatively involved in cell rescue and defense, energy metabolism, transcription and protein regulation, membrane proteins and signal transduction; 18 genes encoding hypothetical proteins with unknown function; five genes without any homology. The expression of ten randomly selected differentially expressed genes confirmed by semi quantitative RT-PCR showed that the expression level in the primordium stage were significantly higher than those in dikaryotic mycelium stage. The results of this study will provide usefu nformation for isolating growth and development related genes so as to help the understanding of the molecular development mechanism of Pleurotus ostreatus.