2.1 Experimental materials
Ridgetail white prawn, Exopalaemon carinicauda, is a lower inverebrate, and our experimental research to this prawn does not involve the issue of animal ethics.
To reduce differences in genetic background, adult individuals were selected from the same family that had been self-bred in the laboratory. Average body length was 4.71±0.37 cm and weight was 1.51±0.24 g. Nine tissues (eyestalk, heart, stomach, gill, intestine, muscle, ovary, hepatopancreas, and ventral cord nerve) were collected from five adult E. carinicauda, and used to analyze the expression characteristics of the pcna gene in the different tissues. The ovaries of E. carinicauda, at different stages of ovarian development (I, II, III, IV, V) were taken to analyze the expression characteristics of pcna in the different stages.
2.2 Extraction of total RNA and synthesis of the first strand of cDNA
A Trizol Total RNA extraction kit (Shanghai, Shangon) was used to extract RNA from the samples. According to the instructions of the UNIQ-1 NA synthesis kit PrimerScriptTM RT Master Mix (TaKaRa), the extracted RNA was reversely transcribed into cDNA and diluted to 50 ng/μL for subsequent experiments. Using part of the RNA as a template, the 5'/3' RACE ready cDNA was synthesized, according to the instructions of the SMARTer RACE 5'/3' kit (TaKaRa), and then stored at -20℃ for later use.
2.3 Cloning the full length of the cDNA sequence of the pcna gene
The partial core sequence of the pcna gene was obtained from the transcriptome database of E. carinicauda established earlier in our laboratory. Based on the core fragment, primers were designed using Primer 5.0 software (Tab. 1), and the full length of the cDNA sequence of the pcna gene was obtained using 5'/3' RACE technology.
Tab. 1. Sequence of primers used in this study
2.4 Sequence analysis of pcna
The open reading frame (ORF) of the pcna gene was analyzed by ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), and its amino acid sequence was deduced by DNAMAN. The obtained amino acid sequences were aligned by BLAST (https://www.ncbi.nlm.nih.gov/), and the theoretical isoelectric points and molecular weights were calculated using DNA Star software. Protein domains were analyzed by Interpro (http://www.ebi.ac.uk/interpro/scan.html), and a phylogenetic tree was constructed using MEGA 6.0 software.
2.5 Analysis of pcna gene expression characteristics
The qRT-PCR primers used for gene expression analysis are shown in Table 1, with 18s RNA as the internal reference. Using E. carinicauda cDNA from nine different tissues and from the ovaries at different developmental stages as a template, a reaction system was designed according to the instructions of the SYBER Premium Ex Taq II kit (TakaRa), and amplification was performed using a real-time PCR instrument (StepOne Plus). The results were obtained using the 2- ΔΔ Ct method of analysis (Schmittgen et al., 2008), with SPSS19.0 software for the one-way ANOVA (p<0.05 indicated a significant difference).
2.6 Recombinant expression and purification of PCNA protein
The pcna gene recombinant expression primers were designed (Table 1), and after amplification, the target fragment and the expression plasmid pET32a were subjected to double enzyme digestion. After recovering the digested product, T4 ligase was used for 2 h to connect it and construct the recombinant plasmid. The successfully constructed recombinant plasmid was transferred into BL21 receptive cells. Trx-His encoded by the empty plasmid pet32a was used as a control protein. The recombinant protein and control protein were purified using a His-tagged protein purification kit (Solube Protein).
2.7 Western blotting
The recombinant protein and purified protein were added to the sample loading buffer and boiled at 95℃ for the SDS-PAGE electrophoresis. Western blot was used to detect His tags to verify the accuracy of the protein.
2.8 Overexpression of PCNA recombinant protein
Purified protein was dissolved in sterile water to achieve a final concentration of 0.15 μg/μL. Healthy adult white shrimp (60 shrimp, 30 shrimp in each group) of uniform size and at gonadal development Stage II were selected, and the PCNA recombinant protein (3 μL) or the Trx-His control protein (3 μL) were injected. To ensure the continuous functioning of the recombinant protein during the experiment, second and third injections were given on the 4th and 8th days after the initial injection. After 0, 1, 3, 5, 7, 9, and 11 d, the shrimp ovaries were removed and the gonadal index (GSI) was calculated:
GSI=wet weight of ovary/body weight of shrimp × 100%
RNA was then extracted and reverse-transcribed into cDNA for qRT-PCR amplification.
2.9 Immunohistochemistry
Ovaries of E. carinicauda were taken and fixed in 4% paraformaldehyde at 4℃ overnight. After being dehydrated in ethanol, samples were embedded in paraffin and sectioned. Sections were deparaffinized, rehydrated in ethanol, and then washed with distilled water. EDTA was added to restore the antigen. A 3% solution of hydrogen peroxide was added to the sections to block endogenous peroxidase. The sections were then washed with PBS, and treated with 3% BSA for 30 minutes. Mouse anti-PCNA polyclonal antibody (1:1000) was added to the sections that were then incubated overnight at 4℃. Sections were washed with PBS, and the second antibody (HRP-labeled goat anti-mouse) was added for 30 minutes . Sections were then washed with PBS, and the dark reaction product was developed with freshly prepared DAB. The color development was timed under a microscope. Hematoxylin was used as a counter stain for 3 minutes, and the sections were then rinsed under tap water, and dehydrated. Sections were viewed under a microscope.
2.10 RNA interference
The Thermo Fisher website (http://.rnaidesigner Thermofisher.com/rnaiexpress/
design.do) was used to design the RNA interference primers (Table 1) and the siRNA was synthesized using a TR102-T7 RNAi transcription kit (Vazyme). Healthy, evenly sized adult shrimp (60 shrimp, 30 in each group) with gonadal development in Stage III were selected for the RNA interference experiments. For the interference group, siRNA was injected into the pericardial cavity (5 μg/g) (Ma et al., 2020), while the control group was injected with normal saline (5 μg/g). At 0, 6, 12, 24, 48, and 72 h after injection, the shrimp ovaries and hepatopancreas were harvested, RNA was extracted and reverse-transcribed into cDNA, and then amplified by qRT-PCR.
2.11 Flow cytometric analysis for ploidy of hepatopancreas
Previous studies showed that the vitellogenin (Vg) is mainly synthesized in follicular cells and hepatopancreas (Li et al., 2010). Here, the hepatopancreas tissue of E. carinicauda is used to analyze the ploidy, because the follicular cells are difficult to be separated from the ovary. After being separated from the corresponding individuals which come respectively from the control group and pcna RNAi group at the time point of interference 24h, the hepatopancreas are cleaned twice with DPBS (PBS without calcium and magnesium). Then, the hepatopancreas was digested with collagenase Ⅱ (2mg/ml) and dispase (0.2mg/ml) for 15min. The digestion solution is first filtered through a 70 μm cell sieve, then filtered through a 40 μm cell sieve, and transferred to a centrifuge tube. Through centrifuging at 500g for 5min, the suspension in the tubes was washed twice with PBS. Add 75% anhydrous ethanol pre cooled at 4℃ to fix the cell suspension and shake slowly to avoid cell clustering. Then, centrifuging the tubes at 1000rpm for 5min to precipitate the cell. Add 3ml of pre cooled PBS in the tubes to wash the precipitated cells. Adds 1ml of DNA staining solution in the tubes, shakes the tubes in a vortex for 5-10 seconds, then incubate them at room temperature in dark for 30min. Ploidy was detected by flow cytometry (FACSVantage, BD, America) at the excitation wavelength of 488nm.