The method of Cdc42 gene accurate tissue location and quantitative analysis in the wheat aphids
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Key Words：Sitobion avenae, Cdc42, in situ hybridization, real time fluorescence quantitative RT-PCR
Abstract： To determine the temporal and tissue-specific profile of Cdc42 gene expression, we performed in situ 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, including the head region and abdomen of both alate and apterous adults and between the compound eyes on the head region. In embryos, staining was mainly evident on the abdomen. There was faint staining on the thorax of alate aphids, 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 apterous aphids. We conclude that in situ hybridization and real-time fluorescence quantitative PCR can determine the location and degree of expression of the Cdc42 gene in wheat aphids