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RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus
Published on: October 16, 2011
Characterization, expression and silencing by RNAi of p53 from Penaeus monodon
Wenting Dai1,2, Lihua Qiu3,4, Chao Zhao1,5
1South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China.
Abstract:
The tumor suppressor p53 is a sequence-specific transcription factor, whose target genes can regulate genomic stability, the cellular response to DNA damage and cell-cycle progression. In the present study, the full-length complementary DNA (cDNA) sequence of p53 gene from Penaeus monodon (Pmp53) was cloned by the technology of rapid amplification of cDNA ends (RACE). The cDNA of Pmp53 was 2239 bp, encoding a protein of 450 amino acids with calculated molecular weight of 50.62 kDa. The temporal expression of Pmp53 in different tissues (ovary, heart, intestine, brain, muscles, stomach and gills) and different developmental stages of ovary was investigated by real-time quantitative PCR (RT-qPCR). The lowest expression level of Pmp53 was observed in the stomach, while the highest expression level was detected in the brain. During the ovary development stages, the expression level of Pmp53 reached the peak at stage III. RNA interference (RNAi) and serotonin (5-hydroxytryptamine, 5-HT) injection experiments were conducted to study the expression profile of Pmp53 and PmCDK2 (cyclin-dependent kinase 2, CDK2). Knocked down of Pmp53 by dsRNA-p53 was sequence-specific and successful. Expression levels of Pmp53 and PmCDK2 in ovary of P. monodon were significantly increased at 12-96 h post 5-HT injection. These results indicate that Pmp53 may be involved in the regulation of ovarian development of P. monodon.
Insights
The tumor suppressor p53 gene in Penaeus monodon (Pmp53) was cloned and its expression studied. Pmp53 plays a role in ovarian development, with levels increasing after serotonin injection.
Area of Science:
- Molecular biology
- Marine biology
- Genetics
Background:
- The p53 tumor suppressor is a key transcription factor regulating genomic stability and DNA damage response.
- Understanding p53's role in invertebrates like Penaeus monodon offers insights into conserved biological pathways.
Purpose of the Study:
- To clone and characterize the p53 gene (Pmp53) from Penaeus monodon.
- To investigate the expression pattern of Pmp53 in various tissues and during ovarian development.
- To explore the relationship between Pmp53, serotonin (5-HT), and ovarian development.
Main Methods:
- Cloning of the full-length Pmp53 cDNA using rapid amplification of cDNA ends (RACE).
- Real-time quantitative PCR (RT-qPCR) for analyzing Pmp53 expression levels.
- RNA interference (RNAi) to knock down Pmp53 expression.
- Serotonin (5-HT) injection to study its effect on Pmp53 and PmCDK2 expression.
Main Results:
- The Pmp53 cDNA sequence was 2239 bp, encoding a 450-amino acid protein.
- Pmp53 exhibited varied expression across tissues, with the highest in the brain and lowest in the stomach.
- Pmp53 expression peaked during stage III of ovarian development and increased significantly post-serotonin injection, along with PmCDK2.
Conclusions:
- Pmp53 is successfully cloned and characterized in Penaeus monodon.
- Pmp53 expression is tissue-specific and dynamically regulated during ovarian development.
- Serotonin significantly upregulates Pmp53 and PmCDK2, suggesting Pmp53's involvement in ovarian development regulation.
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