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

Transcription factor E2F-2 is a regulator of cell cycle. Researchers identified E2F-2 genes from yeasts to humans, but few reports investigated E2F-2 gene from black tiger shrimp. In the present study, we cloned E2F-2 gene from black tiger shrimp (Penaeus monodon). Full-length PmE2F-2 complementary DNA sequence measures 3,189 bp with an open reading frame of 1,371 bp. Complete PmE2F-2 genomic sequence (17,305 bp) of P. monodon contains nine exons, which are separated by eight introns. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that PmE2F-2 is highly expressed in hepatopancreas and ovaries of P. monodon. Highest PmE2F-2 expression levels were observed in stage III ovarian development of P. monodon. PmE2F-2 expression levels were significantly augmented in ovaries of P. monodon after 5-hydroxytryptamine injection and eyestalk ablation. RNA interference experiments were conducted to examine PmE2F-2, PmCDK2, and PmCyclin E expression profiles. PmE2F-2 was successfully knocked down in ovaries and hepatopancreas via double-stranded RNA (dsRNA)–E2F-2 injection. In the same organs, PmE2F-2 expression localization and level were investigated through in situ hybridization, which revealed consistent results with those of qRT-PCR. After dsRNA—E2F-2 injection, gonadosomatic index of shrimp was significantly lower than those following dsRNA—GFP and phosphate-buffered solution injections. Therefore, PmE2F-2 may be involved in ovarian maturation in P. monodon.


Introduction
The E2F transcription factor family was first studied in 1980s as an adenovirus E2 gene promoter activator [1]. Currently, eight E2F family member genes are known, from E2F-1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Total RNA extraction and first-strand cDNA synthesis Total RNA of dissected tissues was extracted using TRIzol reagent (Invitrogen, USA) based on manufacturer's protocol. Total RNA integrity was verified through 1.2% agarose gel electrophoresis, and RNA concentration was determined with NanoDrop-2000 (Thermo Fisher, USA). First-strand cDNA was synthesized from 1 μg of total RNA using PrimeScript Reverse Transcriptase Kit (TaKaRa, Dalian, China).

Genomic DNA isolation
Genomic DNA was prepared from muscle tissues using a previously described standard method [18]. DNA concentration was determined with NanoDrop-2000 (Thermo Fisher, USA).

Characterization of genomic structure and promoter region of PmE2F-2
PmE2F-2 gene sequence was acquired using a PCR-based strategy with genomic DNA as template and full-F and full-R primers (Table 1). Expected DNA fragment was obtained through a previously described standard method [19].

5-HT challenge and eyestalk ablation assay
Neurotransmitters like serotonin (5-HT) is involved in the regulation of ovarian maturation and ovulation. To examine effects of 5-HT on PmE2F-2 mRNA expression, 5-HT creatinine sulfate (Sigma, MO, USA) was dissolved in the sterilized saline solution and made into a 0.25 μmol solution. The first abdominal segment of female shrimp was injected intramuscularly with 50 μL 0.25 μmol 5-HT. Other shrimps were injected with sterilized saline solution (10 mM Tris-HCl at pH 7.5, 400 mM NaCl) at 0 h and were used as control. Ovaries were collected at 0, 6, 12, 24, 48, 72, and 96 h postinjection, snap frozen in liquid nitrogen, and stored at −80˚C.

RNAi assay
The specific primers that contained the T7 promoter site for RNAi experiments were designed using Snap Dragon tools (http://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl). We design the primers (iE2F-2-F/R and iGFP-F/R) for dsRNA synthesis. The Sac I cleavage site was added at the 5-terminus of iE2F-2 / iGFP-F, and the Sal I cleavage site was added at the 5-terminus of iE2F-2 / iGFP-R. Difference in silencing effect was not observed between dsRNAs produced through in vivo bacterial expression and in vitro transcription [13]. Recombinant plasmids (pD7-E2F-2 and pD7-GFP) were established. For in vitro transcription, sense and antisense DNA templates were generated via PCR using pD7-E2F-2 and pD7-GFP recombinant plasmids as templates. dsRNA-E2F-2 and dsRNA-GFP were synthesized in vitro with Transcription T7 Kit (TaKaRa) following manufacturer's instruction. dsRNA was stored at −80˚C.
We chose the stage II of developmental stages of ovarian in P. monodon as the experimental shrimps. P. monodon samples were acclimatized for two days before dsRNA-E2F-2, dsRNA -GFP, and phosphate-buffered solution (PBS) injections. RNAi experiments were performed based on a previously describe standard method [13]. At 0, 6, 24, 48, 72, and 96 h postinjection, ovaries and hepatopancreas were collected and weighed from shrimps injected with dsRNA-GFP, dsRNA-E2F-2, and PBS, snap frozen in liquid nitrogen, and stored at −80˚C. Gonadosomatic index (GSI, ovarian weight / body weight × 100) of each shrimp was calculated.

In situ hybridization
Specific digoxigenin-labeled RNA probes against PmE2F-2 were synthesized by the TaKaRa Company (TaKaRa, Dalian, China). At 24 h postinjection of dsRNA-E2F-2 and dsRNA-GFP, in situ ovary and hepatopancreas hybridization experiments were performed based on a previously described standard method [22].

Sequence analysis
Full-length PmE2F-2 cDNA sequences were analyzed using the Basic Local Alignment Search Tool programs at the National Center for Biotechnology Information (http://blast.ncbi.nlm. nih.gov/Blast.cgi). Complete open reading frame (ORF) regions and amino acid (aa) sequences were analyzed with ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Protein domains were predicted using Simple Modular Architecture Research Tool (http://smart.emblheidelberg.de/). Multiple sequence alignments were created using Clustal W software (http:// www.clustal.org/). Phylogenetic tree was constructed through neighbor-joining method in MEGA 5.03.

Quantitative real-time PCR (qRT-PCR)
qE2F-2-F/R primers (Table 1) were used during qRT-PCR to detect temporal expression of P. monodon. qRT-PCR was performed using SYBR Premix Ex Taq II (TaKaRa, Dalian, China), and its specific methods were carried out based on a previously described standard method [23].

Statistical analysis
Relative mRNA expression levels were examined through one-way analysis of variance (PASW Statistics 18.0; Chicago, IL, USA) in SPSS 22.0. P < 0.05 was considered statistically significant.  Highest identity with different species was noted in functional domain of predicted aa sequence of E2F-2 genes from P. monodon. Fig 3 illustrates a dendrogram depicting evolutionary relationship based on E2F-2 protein similarity of different species. Vertebrate E2F-2 proteins are closely related to each other and converge into one subgroup, whereas P. monodon E2F-2 proteins are clustered with other invertebrate E2F-2s (See S1 Table).
PmE2F-2 mRNA expression during ovarian maturation qRT-PCR was used to detect relative PmE2F-2 mRNA expression levels in different ovarian developmental stages of P. monodon. In ovarian developmental stages III, IV, and V, PmE2F-2 Cloning and functional characterization of transcription factor E2F-2 from black tiger shrimp (Penaeus monodon) mRNA expression levels were significantly higher than those in stages I and II, and PmE2F-2 expression levels were highest in ovarian developmental stage III (P < 0.05, Fig 7). 12, 24, 48, 72, and 96 h compared with that in control group (Fig 8). We also investigated PmE2F-2 expression levels after eyestalk ablation in ovaries of P. monodon. The findings showed that PmE2F-2 expression significantly increased at 3, 6, 12, 24, 48, and 96 h compared with that at 0 h (Fig 9).
PmE2F-2 mRNA expression profile stimulated by dsRNA-E2F-2 We ascertained PmE2F-2 expression levels after dsRNA-RBL injection in ovary and hepatopancreas of P. monodon (after dsRNA-RBL injection, experimental samples were stored in our laboratory). Results demonstrated that from 6 h to 96 h, PmE2F-2 expression in ovary was upregulated in dsRNA-RBL-injected shrimp postinjection relative to control group. However, PmE2F-2 mRNA expression remained unchanged following dsRNA-GFP injection (Fig  10a). In dsRNA-RBL-injected shrimps, PmE2F-2 expression in hepatopancreas was upregulated after 12-48 h; at 96 h postinjection, this expression decreased to nonsignificantly different levels compared with that of the control group (Fig 10b).
We evaluated their expression levels in ovary and hepatopancreas of P. monodon through qRT-PCR assays to determine effects of dsRNA-E2F-2 on PmE2F-2 gene expression. The findings indicate that in ovaries and hepatopancreas, PmE2F-2 expression levels were significantly knocked down 6 h after dsRNA-E2F-2 injection. PmE2F-2 expression levels were inhibited in ovaries and hepatopancreas of dsRNA-E2F-2-injected shrimp after 6-48 h; this expression increased to nonsignificantly different levels compared with that of the control group. PmE2F-2 mRNA expression remained unchanged after dsRNA-GFP injection (Fig 11).
We also investigated PmCDK2 and PmCyclin E expression levels after dsRNA-E2F-2 injection in ovaries and hepatopancreas of P. monodon. After 6 h, PmCDK2 expression was downregulated in ovaries of dsRNA-E2F-2-injected shrimp; after 96 h, this expression increased to nonsignificantly different levels compared with that of the control group (Fig 12a). Compared with that of the control group, PmCDK2 expression was significantly downregulated in hepatopancreas of dsRNA-E2F-2-injected shrimp 6-24 h after injection (Fig 12b). PmCyclin E expression was downregulated in ovaries of dsRNA-E2F-2-injected shrimp 6 h after injection; after 72, this expression increased h to nonsignificantly different levels compared with that of the control group (Fig 13a). In hepatopancreas of dsRNA-E2F-2-injected shrimp, PmCyclin E expression was downregulated 6 h after injection, and it was increased to nonsignificantly different levels after 48 h compared with that of the control group (Fig 13b). Cloning and functional characterization of transcription factor E2F-2 from black tiger shrimp (Penaeus monodon)

In situ hybridization detection of PmE2F-2 expression
Ovarian and hepatopancreatic tissues were selected and subjected to in situ hybridization analysis to determine PmE2F-2 expression site and expression level. Several positive signals were detected in both ovarian and hepatopancreatic tissues following dsRNA-GFP injection ( Fig  14B and 14E), whereas few positive signals were detected after dsRNA-E2F-2 injection ( Fig  14C and 14F). Positive signals were not detected in negative control (Fig 14A and 14D). Based on positive signal quantities, PmE2F-2 expressions were significantly knocked down in ovaries and hepatopancreas after dsRNA-E2F-2 injection compared with that following dsRNA-GFP injection. These results were consistent with those of qRT-PCR test.

GSI detection of ovarian development
To determine the effects of PmE2F-2 gene on ovarian development, we measured ovary weight and body weight of P. monodon. After dsRNA-GFP, dsRNA-E2F-2, and PBS injections, GSI (ovarian weight/body weight × 100) of each shrimp was calculated. After dsRNA-E2F-2 injection, GSIs of shrimp were significantly lower than those after dsRNA-GFP and PBS injections (Fig 15). Cloning and functional characterization of transcription factor E2F-2 from black tiger shrimp (Penaeus monodon) Discussion E2F-2 is a multifunctional regulator involved in several key cellular processes, such as cell cycle control [6] and cell proliferation, differentiation, and apoptosis [7]. Researchers identified E2F-2 genes from yeast to humans, but few reports investigated E2F-2 genes from black tiger shrimp. In the present study, we first reported cloning of E2F-2 gene homologue from P. monodon. In P. monodon, full-length cDNA sequence of E2F-2 gene measures 3,189 bp. Vertebrate E2F-2 proteins are closely related to each other and converge into one subgroup, whereas P. monodon E2F-2 proteins are clustered with other invertebrate E2F-2s. E2F_TDP, Rb_C, and coiled-coil domains are key functional structural domains in mammalian E2F-2 and are conserved in all selected species [12]. These findings suggest that primary E2F-2 protein structure is conserved throughout evolution.
PmE2F-2 gene of P. monodon contains nine exons, which are separated by eight introns; this observation is consistent with the report on H. sapiens E2F-2 (GenBank accession: NC_000001.11). The 5 0 upstream PmE2F-2 sequence contains various transcription regulatory factors, such as GATA, IRF1, HNF-4, SREBP, and SF-1. Further research should focus on mechanism on how these regulatory elements regulate E2F-2 gene transcription.
Tissue distributions of PmE2F-2 were investigated, and qRT-PCR analysis indicated that PmE2F-2 is also constitutively expressed in tissues of healthy black tiger shrimps. Highest PmE2F-2 mRNA level was detected in hepatopancreas, followed by the ovary. Vitellogenin plays a crucial role in ovarian development; it is derived from ovaries, hepatopancreas, or  Relative expression level of PmE2F-2 in the ovary. b. Relative expression level of PmE2F-2 in the hepatopancreas. Ovary and hepatopancreas tissues collected from shrimps injected with dsRNA-RBL were compared with respect to PmE2F-2 mRNA expression (relative to EF-1α) using Students t-tests. Vertical bars represented mean±SD (n = 3). Significant differences from controls were indicated: **P < 0.01, *P < 0.05. https://doi.org/10.1371/journal.pone.0177420.g010 Cloning and functional characterization of transcription factor E2F-2 from black tiger shrimp (Penaeus monodon) adipose tissues of decapod crustaceans [24]. These results demonstrate that PmE2F-2 may be involved in ovarian maturation.
Oocyte development involves various complicated cellular events, which temporally and spatially express differential genes to ensure proper development or storage of transcripts and proteins as maternal factors for early embryogenesis [25,26]. Stage III is the most critical period of ovarian development, and presence of oocytes stimulates accumulation of yolk substances in the cytoplasm [27]. PmE2F-2 expression level was highest in stage III, indicating the important role of PmE2F-2 in ovarian development. This result is consistent with findings of some studies, which showed highest PmCyclin B expression level in stage III [28].
Serotonin (5-HT) was reported to induce ovarian maturation and spawning in black tiger shrimp P. monodon [29]. PmE2F-2 expression of 5-HT-injected shrimp significantly increased at 12, 24, 48, 72, and 96 h compared with that of the control group. PmCDK7 expression level was also augmented following 5-HT injection [17]. However, studies provide limited information on 5-HT regulatory mechanisms in crustaceans. Further investigation is thus needed.
Eyestalk ablation is commonly practiced in crustaceans to induce ovarian maturation in captivity [30]. In our study, we evaluated PmE2F-2 expression levels in ovaries of P. monodon after eyestalk ablation. Results indicated that PmE2F-2 expression significantly increased following eyestalk ablation. This finding is similar to that of a previous study on CDC2 in P. monodon [31] and vitellogenin in Macrobrachium nipponense [32], where expression level also increased after eyestalk ablation. Such result demonstrates that PmE2F-2 may be involved in ovarian maturation.
RNAi was used to clarify latent relationship between PmE2F-2 and ovarian development, particularly the role of PmE2F-2 in ovarian development of P. monodon. Previously published studies described the use of RNAi in illuminating gene functions in shrimps. For example, a study reported that Pmp53 expression knockdown indicates that Pmp53 may significantly influence ovarian development of P. monodon [13]. Silencing of gonad-inhibiting hormone transcripts was performed to establish an efficient RNAi-based technique to stimulate gonadal development in L. vannamei [14]. In the present study, we successfully knocked down PmE2F-2 genes in ovary and hepatopancreas via dsRNA-E2F-2 injection. PmE2F-2 expression of dsRNA-E2F-2-injected shrimp was inhibited after 6-24 h and was then increased after 96 h to nonsignificantly different levels compared with that of the control group. These results are consistent with those of a previous report, which revealed temporary specific inhibition of gene expression through RNAi-based techniques [33]. Several studies reported E2F-2 gene function using RNAi. RNAi-mediated E2F-2 knockdown inhibited human glioblastoma cell tumorigenicity [34]. After 6-72 h of dsRNA-E2F-2 injection, relative PmCDK2 and PmCyclin E expression levels were downregulated in ovaries and hepatopancreas, revealing that PmE2F-2 silencing can decrease PmCDK2 and PmCyclin E expression level of P. monodon. Previous studies often referred to E2F-1, E2F-2, and E2F-3 as "activator" E2Fs because they transcriptionally activate E2F target genes, such as cyclin E and CDK2, that aid cell cycle regulation [10]. Rb functions as a cell cycle repressor through inhibition of E2F transcription factor activity. Hyperphosphorylated Rb releases E2F and promotes expression of genes mediating entry into the S phase [9]. We also determined PmE2F-2 expression levels in ovaries and hepatopancreas of P. monodon following dsRNA-RBL injection. Results demonstrated that in ovaries and hepatopancreas of dsRNA-RBL-injected shrimps, PmE2F-2 expression were upregulated after 6-96 h relative to that of control group. Dai et al. [13] reported that PmCDK2 may be involved in vitellogenin synthesis and ovarian maturation in P. monodon. These findings suggest possible involvement of PmE2F-2 in ovarian maturation.
We studied PmE2F-2 expression site and expression level in ovaries and hepatopancreas via in situ hybridization. Several positive signals were detected in ovary and hepatopancreas following dsRNA-GFP injection. By contrast, only few positive signals were observed after dsRNA-E2F-2 injection. Considering the quantities of positive signals, PmE2F-2 expression levels were significantly knocked down in ovaries and hepatopancreas after dsRNA-E2F-2 injection compared with those following dsRNA-GFP injection. These results were in agreement with those of qRT-PCR test.
In crustaceans, GSI is a gross quantitative indicator of gonad condition and is the simplest way to measure changes in size and weight of this organ relative to total weight of organisms [35]. After dsRNA-E2F-2 injection, GSI of shrimp was significantly lower than those after dsRNA-GFP and PBS injections. These findings demonstrate that PmE2F-2 may play a crucial role in ovarian development.
In conclusion, PmE2F-2 cDNA sequences were cloned and identified. Complete genomic sequence of PmE2F-2 from P. monodon contains nine exons, which are separated by eight introns. PmE2F-2 is highly expressed in hepatopancreas and ovaries and during stage III ovarian development of P. monodon. PmE2F-2 expression levels significantly increased in ovaries of P. monodon following 5-HT injection and eyestalk ablation. PmE2F-2, PmCDK2, and PmCyclin E expression levels were determined after dsRNA-E2F-2 injection to examine their relationship with one another. We investigated PmE2F-2 expression localization and levels in ovaries and hepatopancreas through in situ hybridization, which revealed consistent results with those of qRT-PCR. After dsRNA-E2F-2-injection, GSI of shrimp was considerably lower compared with those after dsRNA-GFP and PBS injections. Findings of this study can improve our understanding of the molecular mechanisms underlying ovarian development in shrimps.