麦蚜Cdc42基因的准确组织定位和定量分析方法研究

The method of Cdc42 gene accurate tissue location and quantitative analysis in the wheat aphids

本文采用原位杂交方法,对Cdc42在麦长管蚜Sitobion avenae(Fabricius)不同组织分布进行定位,结果表明在滴加杂交液的组织切片被特异性的染成棕褐色,而阴性对照不着色;有、无翅蚜的头部和腹部均有特异性着色点,头部的着色点集中在两复眼之间,腹部的着色点几乎全集中在伪胚胎;有翅蚜的胸部几乎全部被着色,而无翅蚜的胸部几乎没有特异着色点。再运用实时荧光定量RT-PCR技术,进行了该基因的相对表达量测定,结果表明麦长管蚜Cdc42基因在不同组织及各虫期均能表达,若蚜期高于成蚜期,有翅蚜高于无翅蚜,且伪胚胎就有较高的转录水平。由此得知,基于原位杂交与实时荧光定量RT-PCR的技术相结合的方法,可有效进行麦蚜Cdc42基因的准确组织定位和定量分析,为Cdc42基因在麦蚜的翅型分化调控上的功能研究奠定了分子检测基础。

To determine the temporal and tissue-specific profile of Cdc42 gene expression,we performed insitu hybridization （ ISH） on different tissues from different nymph instars and adults of alate and apterous English grain aphids,Sitobion avenae （ Fabricius）. After dipping in the hybridization solution,some tissue biopsies stained brown indicating Cdc42 gene expression,but no staining was apparent on the negative control.Staining indicated the presence of the Cdc42 gene transcript in multiple tissues in different life stages,includingthe head region and abdomen of both alate and apterous adults and between the compound eyes on the headregion. In embryos,staining was mainly evident on the abdomen. There was faint staining on the thorax of alateaphids,but hardly any staining in apterous aphids. Tissue and stage specific transcriptive levels were determined by Real Time fluorescence quantitative RT - PCR. This showed that the level of transcription was higher in the first and forth instar than in others,in adults than nymphs in general,and in alate than apterousaphids. We conclude that in situ hybridization and real-time fluorescence quantitative PCR can determine thelocation and degree of expression of the Cdc42 gene in wheat aphids