Expression analysis of a transformer gene in Daphnia pulex after RNAi

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• 作者：C. Y. Guo ; P. Chen ; M. M. Zhang ; J. J. Ning ; C. L. Wang ; D. L. Wang…
• 关键词：RNAi ; RT ; PCR ; Western blotting ; reproductive transformation ; parthogenesis ; cladocerans
• 刊名：Molecular Biology
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：50
• 期：5
• 页码：748-754
• 全文大小：577 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Life Sciences
  Biochemistry
  Human Genetics
  Russian Library of Science
  
• 出版者：MAIK Nauka/Interperiodica distributed exclusively by Springer Science+Business Media LLC.
• ISSN：1608-3245
• 卷排序：50

文摘

In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.