The Prepropalustrin-2CE2 and Preprobrevinin-2CE3 Gene from Rana Chensinensis: Gene Expression, Genomic Organization, and Functional Analysis of the Promoter Activity

Background: Palustrin-2CE2 and brevinin-2CE3 are antimicrobial peptides from Rana chensinensis. In R. chensinensis tadpoles, the expression of prepropalustrin-2CE2 and preprobrevinin-2CE3 increased with the developmental stage. In addition, the expression of the two genes was dramatically upregulated with stimulation by Escherichia coli, Staphylococcus aureus, and the chemical lipopolysaccharide (LPS). The genomic organization of the two antimicrobial peptide genes was confirmed. Both prepropalustrin-2CE2 and preprobrevinin-2CE3 contain three exons separated by two large introns. Additionally, several presumed transcription factor binding sites were identified in the promoter sequence. Functional analysis of the promoter was performed using a luciferase reporter system, and further confirmed by yeast one-hybrid experiment and EMSA assay. The results indicated that the transcription factors NF-κB and RelA are involved in regulating the expression of prepropalustrin-2CE2 and preprobrevinin-2CE3. As amphibian populations decline globally, this study provides new data demonstrating how frogs defend against pathogens from the environment by regulating AMP expression. For amphibians, antimicrobial peptides are innate immune molecules that resist adverse external environmental stimuli. However, the regulation mechanism of antimicrobial peptide gene expression in frogs is still unclear. Objective: The two antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, are produced under external stimulation in Rana chensinensis. Using this model, we analyzed the gene structure and regulatory elements of the two antimicrobial peptide genes and explored the regulatory effects of related transcription factors on the two genes. Method: Different stimuli such as E. coli, S. aureus, and chemical substance lipopolysaccharide (LPS) were applied to Rana chensinensis tadpoles at different developmental stages, and antimicrobial peptide expression levels were detected by RT-PCR. Bioinformatics analysis and 5'-RACE and genome walking technologies were employed to analyze the genome structure and promoter region of the antimicrobial peptide genes. With dual-luciferase reporter gene assays, yeast one-hybrid experiment and EMSA assays, we assessed the regulatory effect of the endogenous regulators of the cell on the antimicrobial peptide promoter. Results: The transcription levels of prepropalustrin-2CE2 and preprobrevinin-2CE3 were significantly upregulated after different stimulations. Genomic structure analysis showed that both genes contained three exons and two introns. Promoter analysis indicated that there are binding sites for regulatory factors of the NF-κB family in the promoter region, and experiments showed that endogenous NF-κB family regulatory factors in frog cells activate the promoters of the antimicrobial peptide genes. Yeast one-hybrid experiment and EMSA assay demonstrated that RelA and NF-κB1 might interact with specific motifs in the prepropalustrin-2CE2 promoter. Conclusion: In this paper, we found that the gene expression levels of the antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, in R. chensinensis will increase under environmental stimuli, and we verified that the changes in gene expression levels are affected by the transcription factors RelA and NF-κB1. The yeast one-hybrid experiment and EMSA assay confirmed that RelA and NF-κB1 could directly interact with the frog antimicrobial peptide gene promoter, providing new data for the regulatory mechanism of antimicrobial peptides in response to environmental stimuli.

Determination and Chemometrics-Assisted Comparative Analysis of Active Components in Different Tissue of Rana chensinensis

In this study, the chemical composition of different tissues of Rana temporaria chensinensis David derived from the same individual was analyzed by comparative approach. First, pre-column derivatization combined with high performance liquid chromatography (HPLC) was established to determine the content of 1-methyl hydantoin in samples, which used S1–S5 samples. The results indicated that 1-methyl hydantoin was determined in Oviductus Ranae (OR), Rana chensinensis ovum (RCO), Rana chensinensis meat (RCM), and Rana chensinensis skin (RCS), except for Rana chensinensis bone (RCB). Moreover, the content of it in RCS was the highest. In addition, the contents of six polyunsaturated fatty acids (PUFAs) in different tissues of Rana chensinensis were measured by HPLC, including eicosapentaenoic acid (EPA), α-linolenic acid (ALA), docosahexaenoic acid (DHA), arachidonic acid (ARA), linoleic acid (LA) and oleic acid (OA). The results indicated that OR, RCO, RCM, RCS, and RCB all contained the above six PUFAs. With the aid of chemometrics methods, the results of principal component analysis (PCA), hierarchical cluster analysis (HCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) combined with the sequencing results of the total PUFAs content of each sample, showed that different tissues of Rana chensinensis could be divided into four categories, and the RCO sample was divided into one category because of the highest PUFAs content, which was a good source of PUFA. For comparison, OR and other tissue from the perspective of PUFAs, we also established OPLS-DA models of them. It could be found that the RCM was the most similar to the OR in the diversity and content of PUFAs. This study provided a theoretical basis for the further development and utilization of RCO, RCM, RCS, and RCB as by-products of OR.

Isolation and Physicochemical Characterization of Melanin from Ova of Rana chensinensis

Melanin was widely used in the food, pharmaceutical, and chemical industries. In this study, melanin was isolated from the ova of Rana chensinensis (OORC) by means of enzymatic hydrolysis assisted with NaOH. Infrared spectrometry, elemental analyzer, and UV-VIS absorption spectroscopy were performed to explore optimal protein extraction methods and physicochemical characterization of extracted OORC melanin. The results showed that OORC melanin was mainly composed of amino, hydroxy, C=C, C=O, CH2, CH, and aromatic ring groups. The S:N ratio in the OORC melanin was 0.05, consisting mostly of eumelanin and a little protein. The optimal protein extraction method was 0.66 mol/L NaOH, 40 °C for 87 min, and the suitable ratio of NaOH solution to raw material was 20.7:1. This research was aimed at providing a reference for the development and utilization of melanin.

Rana chensinensis Ovum Oil Based on CO2 Supercritical Fluid Extraction: Response Surface Methodology Optimization and Unsaturated Fatty Acid Ingredient Analysis

Rana chensinensis ovum oil (RCOO) is an emerging source of unsaturated fatty acids (UFAs), but it is lacking in green and efficient extraction methods. In this work, using the response surface strategy, we developed a green and efficient CO2 supercritical fluid extraction (CO2-SFE) technology for RCOO. The response surface methodology (RSM), based on the Box–Behnken Design (BBD), was used to investigate the influence of four independent factors (pressure, flow, temperature, and time) on the yield of RCOO in the CO2-SFE process, and UPLC-ESI-Q-TOP-MS and HPLC were used to identify and analyze the principal UFA components of RCOO. According to the BBD response surface model, the optimal CO2-SFE condition of RCOO was pressure 29 MPa, flow 82 L/h, temperature 50 °C, and time 132 min, and the corresponding predicted optimal yield was 13.61%. The actual optimal yield obtained from the model verification was 13.29 ± 0.37%, and the average error with the predicted value was 0.38 ± 0.27%. The six principal UFAs identified in RCOO included eicosapentaenoic acid (EPA), α-linolenic acid (ALA), docosahexaenoic acid (DHA), arachidonic acid (ARA), linoleic acid (LA), and oleic acid (OA), which were important biologically active ingredients in RCOO. Pearson correlation analysis showed that the yield of these UFAs was closely related to the yield of RCOO (the correlation coefficients were greater than 0.9). Therefore, under optimal conditions, the yield of RCOO and principal UFAs always reached the optimal value at the same time. Based on the above results, this work realized the optimization of CO2-SFE green extraction process and the confirmation of principal bioactive ingredients of the extract, which laid a foundation for the green production of RCOO.