Characterization, expression and silencing by RNAi of p53 from Penaeus monodon

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.

Molecular cloning, characterization and expression analysis of a novel PDRG1 gene from black tiger shrimp (Penaeus monodon)

“…In ovary, the silencing efficiency of Pmp53 at 12, 24, 48 and 72 h of dsRNA-p53 post-injection were 65.86, 85.35, 64.06 and 25.45%, respectively, and in hepatopancreas, the silencing efficiency from 12, 24, 48, 72 and 96 h post injection were 37.70, 39.29, 64.24, 88.67 and 20.37%, respectively. Detailed data on Pmp53 gene silencing have been published in our previous study (Dai et al ., 2016). After Pmp53 was successfully silenced, the PmPDRG1 mRNA expression pattern was analyzed by RT-qPCR assays in ovary and hepatopancreas.…”

“…As stage III of ovary development is marked by massive cell proliferation and the presence of oocytes that have accumulated yolk substances in the cytoplasm (Huang et al ., 2006), the results indicate that PmPDRG1 may be related to the oogenesis stage of ovarian development. In our previous study, we found that Pmp53 plays an important role in the development and maturation of the ovaries in P. monodon (Dai et al ., 2016). To now study the relationship between Pmp53 and PmPDRG1 we successfully silenced the Pmp53 gene (Dai et al ., 2016) causing an up-regulation of the relative expression of PmPDRG1 both in the ovary and hepatopancreas, indicating that Pmp53 could down-regulate PmPDRG1 transcript levels.…”

“…In our previous study, we found that Pmp53 plays an important role in the development and maturation of the ovaries in P. monodon (Dai et al ., 2016). To now study the relationship between Pmp53 and PmPDRG1 we successfully silenced the Pmp53 gene (Dai et al ., 2016) causing an up-regulation of the relative expression of PmPDRG1 both in the ovary and hepatopancreas, indicating that Pmp53 could down-regulate PmPDRG1 transcript levels. The molecular regulatory mechanisms, however, still need to be further studied.…”

“…Therefore, it is imperative to explore alternative technologies to eyestalk ablation and to understand the molecular mechanisms that control the development and maturation of ovaries/oocytes (Hiransuchalert et al ., 2013; Phinyo et al ., 2014). In our previous study, we found that Pmp53 plays an important role in the development and maturation of the ovaries in P. monodon (Dai et al ., 2016). In the present study, we silenced Pmp53 to investigate its relationship with PmPDRG1 and the role that PmPDRG1 may play in the ovarian development of P. monodon .…”

Genomic structure, expression pattern, and functional characterization of transcription factor E2F-2 from black tiger shrimp (Penaeus monodon)

“…For example, knockdown of Pmp53 expression indicates that Pmp53 may play an important role in ovarian development of P . monodon [13]. Gonad-inhibiting hormone transcripts are silenced to exploit efficient RNAi-based techniques, which stimulate gonadal development and spawning in Litopenaeus vannamei [14].…”