牙鲆(Paralichthys olivaceus)抗缪勒氏管激素Ⅱ型受体基因(amhr2)表达特征分析及功能初探

抗缪勒氏管激素Ⅱ型受体(anti-Müllerian hormone receptorⅡ,Amhr2)是抗缪勒氏管激素(anti-Müllerian hormone,Amh)的特异受体。amhr2基因在鱼类性腺分化和发育中发挥重要作用,更决定了有的鱼种的性别,然而,相关功能研究较为有限。本研究旨在明晰amhr2在我国重要海水养殖鱼类—牙鲆(Paralichthys olivaceus)性腺分化和发育过程中的表达特征,并初步探究其功能。首先,克隆了牙鲆amhr2-CDS序列,共1536bp,编码511个氨基酸。系统发育分析显示牙鲆Amhr2近C端较为保守,与其他硬骨鱼类聚为一枝,并与上游sp1以及下游的prr13和PCBP2在基因组中共定位。蛋白结构分析显示,牙鲆Amhr2包含信号肽、跨膜结构域和保守的酪氨酸蛋白激酶结构域。进而,利用实时荧光定量PCR(q PCR)分析表明,牙鲆amhr2主要表达于性腺,且在精巢中的表达极显著高于卵巢(P<0.01),其在I-V期精巢中持续高表达,而在卵巢中仅在I期高表达,后显著下降(P<0.05)。性腺分化期基因的表达,是对雌核发育组(对照组,20±0.5℃,100%雌性)与雌核发育高温诱导组(HT组,28±0.5℃,100%雄性)鱼苗进行检测的,amhr2在对照组全长(totallength,TL)2cm的实验鱼性腺中表达最高,后逐渐下降;而HT组实验鱼性腺中的表达呈现先上升后下降的趋势,并在精巢开始分化的6cm TL时表达最高。利用Hela细胞进行亚细胞定位分析发现,Amhr2与其配体Amh均定位于细胞质。同时,通过原核表达重组牙鲆Amh,并用其孵育离体性腺组织,q PCR分析显示精巢中amhr2的表达没有显著变化,但卵巢中表达显著上升(P<0.05)。进一步通过双荧光素酶报告分析表明,Amh和Amhr2共转染能够显著抑制雌激素合成的关键芳香化酶基因cyp19a的表达(P<0.01)。综上所述,牙鲆amhr2主要表达于精巢,但在不同性腺发育时期表达不同,且其可能与Amh共同影响cyp19a的转录,对雄性表型形成的启动和性腺发育起作用。

Transcriptome Analysis Reveals the Regulation Role of miR-144-5p in Intestinal Immunity of Japanese Flounder(Paralichthys olivaceus)

MicroRNAs(miRNAs),22-nucleotide-long micromanagers that guide the post-transcriptional regulation of a wide range of target genes,can theoretically be used as a diagnostic or therapeutic target for inflammatory reaction.In fish,miR-144-5p expression varies dramatically in response to the different bacterial infections and can regulate immunity-related genes to reduce the occurrence of inflammation.In this research,the regulation function of miR-144-5p to the intestinal innate immunity was udied in flounder Paralichthys olivaceus.The flounders were interfered by 2μg g^(-1) miR-144-5p antagomir and their tissues(intestine,liver and spleen)were harvested from the fish at 12 h post-injection.More than 60 million high-quality reads were collected.At 24 hours after miR-144-5p or miR-NC interference,a total of 2704 and 1823 different-expresion genes(DEGs)were identified in comparison with control group,respectively.The DEGs were enriched in a variety of immunity-related signaling pathways,including NOD-like receptor,Wnt and Toll-like receptor signaling pathways,according to GO and KEGG analyses.A total of 503 highly interacting DEGs engaged in 33 immunity-related signaling pathways were discovered using KEGG analyses.Additionally,5 hub genes were found by protein-protein interaction networks,which formed an intricate immune regulation network.Meanwhile,these hub genes were mostly involved in focal adhesion,Wnt signaling pathway,as well as the Intestinal Immune Network for IgT(IgA)Production Pathway.In conclusion,the loss of miR-144-5p can affect immunity-related genes and downstream signaling pathways.Our findings suggest that miR-144-5p is a modulator of gene networks and signaling pathways associated with intestinal innate immunity.

显微注射抗冻剂对牙鲆(Paralichthys olivaceus)胚胎毒性及冷敏感性的影响

在鱼类胚胎冷冻保存中,由于胚胎体积大、卵膜具多层结构,并且通透性差,传统的平衡法无法使抗冻剂足够、有效的进入胚胎内部起到保护作用。为弥补了普通平衡法的不足,此研究利用显微注射技术,将抗冻剂直接注射到鱼类胚胎卵黄囊内,提高了抗冻剂对胚胎的保护程度。此研究以牙鲆胚胎为试验材料,对显微注射抗冻剂种类、抗冻剂的注射剂量、注射浓度、以及抗冻剂注射后胚胎的冷敏感性影响进行了筛选。结果表明:牙鲆胚胎较适宜的显微注射剂量为750pl;几种抗冻剂注射牙鲆胚胎后的毒性大小相对排列为PVP>蔗糖,DMSO>MeOH>PG,其中注射PG获得的胚胎成活率和孵化率最高,注射PVP获得的胚胎成活率和孵化率最低,而PG与MeOH组成混合抗冻剂PM毒性更低获得的胚胎成活率和孵化率更高。而任何一种抗冻剂均随着浓度的增加其毒性随之增加;另外,在冷敏感试验中,显微注射6mol/L的PM的胚胎胚胎,应用程序化法处理,以2℃/min的速率由室温降至-20℃,平衡10min后解冻处理,发现注射PM的胚胎低温处理后成活率为(25.07±1.57)%,而6mol/的PM五步平衡法同步处理的对照组胚胎成活率为(20.88±2.84)%,说明显微注射的胚胎冷敏感性一定降低。

台湾海峡野生牙鲆(Paralichthys olivaceus)亲鱼驯养和促熟产卵的探讨

本文研究了产自台湾海峡的野生牙鲆亲鱼的驯养、促熟和产卵。结果表明 ,活的鲻鱼苗可用于牙鲆的诱食 ;当水温达到 1 6 .2～ 1 9.5 ℃时 ,牙鲆可自然产卵 ,产卵盛期为 3月中旬至 4月上旬 ,4月份受精率高达 86 %。在驯养过程中亲鱼未发生死亡。

Estimates of genetic and phenotypic parameters for weight and length in Paralichthys olivaceus(Temminck et Schlegel)

The complete diallel cross design was employed to estimate the genetic parameters of the growth of Japanese flounder （Paralichthys olivaceus）. A total of 60 full-sib families were cultivated and two growth-related traits, body weight （BW） and body length （BL）, were examined at average 2, 3 and 8 months of age respectively, with 1 800 individuals measured in each age group （30 per family）. Based on the additive-dominance-maternal-effect genetic analysis model, the restricted maximum likelihood approach was used to estimate various （co）variance components. The results showed that narrow-sense heritability estimates of BW and BL were respectively： 0.29 ± 0.01 and 0.22 ± 0.02 at 2 months of age, 0.32 ± 0.02 and 0.30 ± 0.04 at 3 months of age, 0.48 ± 0.04 and 0.40 ± 0.05 at 8 months of age; broad-sense heritability estimates were respectively： 0.44 ± 0.02 and 0.54 ± 0.04 at 2 months of age, 0.35 ± 0.01 and 0.36 ± 0.03 at 3 months of age, 0.49 ± 0.03 and 0.45 ± 0.04. All heritabilities were statistically significant （P 0.05）. Additive genetic correlations between BW and BL at 2, 3 and 8 months of age were consistently positive and highly significant （P 0.01）： 0.93 ± 0.02, 0.95 ± 0.03 and 0.92 ± 0.03 respectively. Maternal effect was significant （P 0.05） only at 2 months of age, and was not detected at 3 and 8 months of age. According to the heritability estimates, the mass selection strategy should be efficient for the breeding of Japanese flounder.

Identification of candidate piRNAs in the gonads of Paralichthys olivaceus (Japanese flounder)

Piwi-interacting RNA （piRNA） plays an important role in the gonadal development and maintenance of Teleostei. In this study, piRNA libraries derived from the adult gonads of Japanese flounder （Paralichthys ofivaceus） were generated using next-generation sequencing technology. Using zebrafish piRNAs as a reference, 5 865 unique candidate piRNAs were identified; 289 candidate piRNA clusters （PRCs） were generated from the above piRNAs. Among the isolated candidate PRCs, a total of 38 ovary-specific, 45 ovary-bias, 24 testis-specific, and 131 testis-bias PRCs were found. The relative expression levels of seven PRCs were validated through quantitative reverse transcription-polymerase chain reaction. The results of this study will help facilitate exploration of the development and maintenance of the phenotypic sex mechanism in P. olivaceus.

Quantitative trait loci detection of E dwardsiella tarda resistance in Japanese flounder Paralichthys olivaceus using bulked segregant analysis

In recent years, Edwardsiella tarda has become one of the most deadly pathogens of Japanese fl ounder( Paralichthys olivaceus), causing serious annual losses in commercial production. In contrast to the rapid advances in the aquaculture of P. o livaceus, the study of E. tarda resistance-related markers has lagged behind, hindering the development of a disease-resistant strain. Thus, a marker-trait association analysis was initiated, combining bulked segregant analysis(BSA) and quantitative trait loci(QTL) mapping. Based on 180 microsatellite loci across all chromosomes, 106 individuals from the F1333(♀: F0768 ×♂: F0915)(Nomenclature rule: F+year+family number) were used to detect simple sequence repeats(SSRs) and QTLs associated with E. tarda resistance. After a genomic scan, three markers(Scaffold 404-21589, Scaffold 404-21594 and Scaffold 270-13812) from the same linkage group(LG)-1 exhibited a signifi cant difference between DNA, pooled/bulked from the resistant and susceptible groups( P <0.001). Therefore, 106 individuals were genotyped using all the SSR markers in LG1 by single marker analysis. Two different analytical models were then employed to detect SSR markers with different levels of signifi cance in LG1, where 17 and 18 SSR markers were identifi ed, respectively. Each model found three resistance-related QTLs by composite interval mapping(CIM). These six QTLs, designated q E1–6, explained 16.0%–89.5% of the phenotypic variance. Two of the QTLs, q E-2 and q E-4, were located at the 66.7 c M region, which was considered a major candidate region for E. tarda resistance. This study will provide valuable data for further investigations of E. tarda resistance genes and facilitate the selective breeding of disease-resistant Japanese fl ounder in the future.

Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis.A new Sertoli cell line(POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages.Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P.olivaceus.Cells were optimally maintained at 25℃ in DMEM/F12 medium supplemented with fetal bovine serum,basic fibroblast growth factor,and epidermal growth factor.The growth curve of POSC showed a typical "S" shape.Chromosome analysis revealed that the cell line possessed the normal P.olivaceus diploid karyotype of 2n=48t.POSC expressed dmrt1 but not vasa,which was detected using RT-PCR and sequencing.Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL.Therefore,POSC appeared to consist of testicular Sertoli cells.Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid,with the transfection efficiency reaching 10%.This research not only offers an ideal model for further gene expression and regulation studies on P.olivaceus,but also serves as valuable material in studying fish spermatogenesis,Sertoli cell-germ cell interactions,and the mechanism of growth and development of testis.

Genetic analysis of meio-and mito-gynogenetic stocks of Paralichthys olivaceus with microsatellite markers

Twenty microsatellite markers （ Pol, Po13, Po33, Po35, Po42, Po48, Po56, Po89, Po91, kop6, kop7, kop8, kop12, kop15, kop18, kop21, kop22, kop23, kop26, Po-str1 ） were used to assess the meiogynogenetic and mitogynogenetic stocks of the lefteyed flounder, Paralichthys olivaceus, which were derived from single pair crossing. Twelve of the 20 loci utilized showed heterozygosity in the female and were mapped in relation to their centromeres in the meiogynogenetic diploid flounder. Microsatellitecentromere map distance, calculated under the assumption of complete interference, ranged from 15. 8 cM for kop22 to 50 cM for Po13, Po56 and Po89. Excluding the kop22, the heterozygosities of the rest of the loci were close to 100% , suggesting the occurrence of near complete interference on the chromosome arms that carried these loci. In the mitogynogenetic diploid flounder, each individual showed exact homozygousity and the segregation profiles did not deviate from the Mendelian 1：1 pattern. The results indicated that there was no lethal gene linked with the loci analyzed. Such high interference accounted for the high recombination rates and large map distances. The Po13 and Po56 loci, Po91 and kopl8 loci, kopl5 and kop21 loci are tightly linked on the same chromosome arm in pairs.

Studies on the edwardsiellosis and characterization of pathogenic bacteria from diseased flounder (Paralichthys olivaceus L.) and turbot (Scophthalmus maximus L.)

Edwardsiellosis of flounder and turbot occurring in different mariculture farms during 2001～ 2004 was examined, including the conditions of disease occurrence, clinical signs and pathological changes. The results showed that all diseased fishes expressed bacterial septicaemia. A total of 148 strains were identified using a combination of traditional physiological and biochemical tests and partial 16S rRNA gene analysis. In addition, the mole fraction G ＋ C ratio of the DNA of representative strain of isolates and serum homology were detected, and pathogenicity tests of isolates were conducted by experimental infection. The results revealed that 148 strains were identified as E. tarda of genus Edwardsiella, all the isolates are of serologic similarity, and have strong pathogenicity to flounder and turbot.

Expressions of Toll Like Receptor(TLR)Genes in Paralichthys olivaceus After Induced by Different Extracts of Edwardsiella tarda

Toll like receptors(TLRs)are the main innate immune‘pattern recognition receptors’of animals,which play a central role in host cell recognition and responses to invasive pathogens,particularly common structures of microbial pathogens.In this study,the gene expression profiles of TLRs in the spleen,head kidney,gill,small intestine,liver,muscle,and heart of healthy Paralichthys olivaceus were detected by real-time quantitative PCR(qPCR).The TLR family members were widely expressed in different tissues with different basic expression profiles.The highest expressions of TLR1,5m,7,8,9,14,and 21 were found in the spleen;the highest expressions of TLR3 and TLR21 were found in the gill;the highest expressions of TLR2 and 5s were found in the small intestine.The second highest expressions of TLR3,7,and 8 were found in small intestine.The gene expression profiles of TLRs stimulated with Edwardsiella tarda DNA,RNA,and lipopolysaccharide(LPS)were also detected in spleen,head kidney and gill.TLR9 and TLR21 were sensitive to E.tarda DNA;TLR 8 and TLR21 were sensitive to E.tarda RNA;and TLR1 and TLR14 were sensitive to E.tarda LPS.The expressions of the other TLR genes showed no significant changes.The results imply that the expressions of these TLR genes in P.olivaceus are differently regulated in the whole body and play important roles in the immune response against E.tarda infection.

Molecular Characterization, Expression Profiles, and Immunostimulation Responses of TRAF6 and TAK1 in Japanese Flounder(Paralichthys olivaceus)

Tumor necrosis factor receptor-associated factor 6(TRAF6) and transforming growth factor-β-activated kinase 1(TAK1) are two important adaptor molecules in Toll-like receptor(TLR) signaling pathway. In this study, TRAF6(PoTRAF6) and TAK1(PoTAK1) were cloned and characterized in Japanese flounder(Paralichthys olivaceus). The full-length cDNA sequence of Po TRAF6 is 1953 bp, with an open reading frame(ORF) of 1713 bp encoding a putative protein of 570 amino acids. PoTRAF6 contains one really interesting new gene(RING) domain, two zinc fingers, one coiled-coil region, and one meprin and TRAF homology(MATH) domain, which shows a high similarity to TRAF6 s in other species. The full-length PoTAK1 cDNA sequence is 2086 bp, with an ORF of 1728 bp that encodes a putative protein of 575 amino acids. PoTAK1 contains a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. The promoter regions of PoTRAF6 and PoTAK1 were also analyzed to predict several potential transcription factor-binding sites. In addition, the expression patterns of these two genes were examined in developmental stages, different tissues, and challenged samples. PoTRAF6 and PoTAK1 were expressed during the whole developmental stages, and the highest expressions were in intestine and heart, respectively. In challenged embryonic cells with LPS, CpG ODN, and poly I:C, the expressions of PoTRAF6 and PoTAK1 were both up-regulated significantly. These results suggest that PoTRAF6 and Po TAK1 play crucial roles in immune responses and may be involved in the developmental process of Japanese flounder.

Cytogenetic characterization of olive flounder Paralichthys olivaceus: DNA content, karyotype, AgNORs and location of major ribosomal genes

A cytogenetic analysis of Paralichthys olivaceus was carried out using the flow cytometry method for DNA content, silver staining for the nucleolus organizer region （AgNORs） identification and one-color fluorescence in situ hybridization （FISH） for chromosomal mapping of major ribosomal genes. Nuclear DNA content was estimated by flow cytometry method using Gallus domesticus erythrocytes as the internal reference standard. The C-value of this species was （0.737±0.024） pg, and the DNA contents of each chromosome were estimated to be 16.51 Mb to 39.50 Mb after paired according to the average relative length. The FISH probe was made by PCR amplification of a DNA fragment containing internal transcribed spacers ITS1 between 18S and 5.8S ribosomal RNA gene, and labeled by PCR incorporation of bio-16-dUTP. FISH signals and AgNORs were both located on the secondary constrictions of chromosome 1. These results will provide a better understanding of the cytogenetic information of this species and would help for further research of the karyotype evolution in the order Pleuronectiformes.

Effects of florfenicol exposure on growth,development and antioxidant capacity of flounder Paralichthys olivaceus larvae at different developmental stages

Antibiotics are widespread in various environmental media,and may pose a potential threat to aquatic ecosystems and non-target aquatic organisms.Florfenicol(FLO)is one of the most commonly used antibiotics in aquaculture,and extensively used to substitute chloramphenicol with its strong sterilization and low adverse ef fect.In this study,flounder Paralichthys olivaceus,an important economic fish species in seawater was used as an experimental subject.Five exposure concentrations of FLO(including environmentrelated concentrations)were set at 0,0.01,0.1,1,and 10 mg/L.Ef fects of FLO exposure for 168 h on growth and development,motor behavior,antioxidant enzyme activity,malondialdehyde(MDA)content,and thyroid hormone level of P.olivaceus larvae were studied in pre-larvae(1 dpf)and post-larvae(20 dpf).The results show that the short-term FLO exposure could promote the larvae growth to some degrees,but inhibit them as the exposure time prolonged.For pre-larvae,FLO at 0.01 mg/L could stimulate the motor nerve system and increase the swimming ability,but inhibited it at 1 mg/L.With the increasing dosage of FLO,the superoxide dismutase(SOD)and MDA contents were elevated,reaching the maximum in the 1 mg/L FLO group.The pre-larvae were more sensitive than the post-larvae to FLO in the environment,and the growth and immune resistance could be damaged with long exposure.Post-larvae were more tolerant to external pollutants,FLO at 1 mg/L could promote the motor behavior and reduce SOD and MDA contents.Therefore,FLO can be used as an antibiotic at a proper concentration but as a drug to prevent disease in a long-term way.

Compensatory Growth of Juvenile Brown Flounder Paralichthys olivaceus Following Low Temperature Treatment for Different Periods

We investigated the effects of low temperature(8.5℃) on the growth and feeding rates and feed conversion efficiency of juvenile P.olivaceus with an average initial weight of 3.87 ± 0.06 g(mean ± SE).Fish were exposed to 8.5℃ for 0(control),1,2,3 and 4 weeks,and then to 20℃ for 10,9,8,7 and 6 weeks,respectively.Low temperature clearly led to growth depression.The weight of fish exposed to low temperature for 1 week was restored to that of control,while that of fish exposed to low temperature longer was significantly decreased(P < 0.05).During the entire low-temperature period,specific growth rate,feeding rate and feed conversion efficiency of the fish were significantly lower(P < 0.05) than those of control,while in the whole recovery period,specific growth and average feeding rate were markedly higher(P < 0.05) than those of control.At the end of experiment,only the feeding rate of the fish exposed to low temperature for 1 week was not significantly different from that of control(P > 0.05).Feeding rate and feed conversion efficiency were reduced at low temperature in juvenile P.olivaceus.The compensatory growth of juvenile P.olivaceus may therefore be attributed to the improvement of feeding rate.Our results suggested that growth depression occurs when juvenile P.olivaceus are exposed to low temperature for more than one week.

GATA4 Is a Transcriptional Regulator of SOX2 in Japanese Flounder(Paralichthys olivaceus)

GATA4,as a member of the GATA transcription factor family,plays a significant role in lineage specification and trans-differentiation of cells by regulating target gene expression.In this study,the potential transcription factor binding sites in the pro-moter of Paralichthys olivaceus sox2 gene were analyzed,and a GATA4-binding motif was identified.A cleaved amplified poly-morphic sequence-based binding assay experiment was performed,and the results showed a significant binding process between the Homo sapiens GATA4 recombinant protein and the DNA fragment of P.olivaceus sox2 promoter.Luciferase reporter assay revealed that GATA4 could improve the activity of the P.olivaceus sox2 promoter.Overexpression of P.olivaceus GATA4 could also increase the sox2 mRNA expression level in flounder brain cells.Overall,our results indicate that sox2 is a target gene of GATA4 in P.oliva-ceus.Sox2 is implicated in the maintenance of stem cell pluripotency and can reprogram differentiated cells to iPS cells.In addition,sox2 is involved in the development of the central nervous system(CNS).Therefore,this study may provide a reference for exploring the function of P.olivaceus GATA4 in the regulation of pluripotent stem cells and the development of the CNS.

Establishment and Characterization of Four Long-Term Cultures of Neural Stem/Progenitor Cell Lines from the Japanese Flounder Paralichthys olivaceus

Neurogenesis is an important progress wherein the neural stem cells(NSCs) differentiate into functional neurons under conductive conditions. Neurogenesis occurs continuously in different areas of the central neural system in adult teleosts compared with adult mammals. Therefore, NSC cell lines must be established to offer a valuable in vitro system for studies on neurogenesis and other related functions. In this study, four cell lines designated as PoB1, PoB2, PoBf and PoBh were established from the brain of the Japanese flounder Paralichthys olivaceus. The cell lines were sub-cultured over 150 times and still grew well in DMEM/F12 medium at 24℃. PoB1, PoB2, PoBf and PoBh were identified as neural stem/progenitor cell lines on the basis of the mRNA expression of nestin and/or aldh111 or slc6 a4 and the formation of neurospheres. The cells transfected with the pEGFP-C1 plasmid showed fluorescent signals with distinct reagent dependencies. The established cell lines from the brain of P. olivaceus offer a valuable system in vitro for the study of neurogenesis, fish neural regulation and endocrinology-related functions.

Molecular Characterization, Expression Pattern and Transcriptional Regulation of Figla During Gonad Development in Japanese Founder (Paralichthys olivaceus)

The factor in the germline alpha(figla), as a member of the basic helix-loop-helix family, has been reported to be involved in ovary development in mammals and teleosts. However, the regulatory mechanisms of figla in teleosts remain unclear. Here,figla in P. olivaceus(Pofigla) was characterized with encoding a 202 amino acid protein that contains a conserved basic region and helix-loop-helix(HLH) domain. Amino acids alignment and synteny analysis revealed that Pofigla was conserved with the orthologous gene sequences in other vertebrates. The results of qRT-PCR showed Pofigla was maternally inherited during embryonic development. For tissue distribution, Pofigla showed a sexually dimorphic gene expression in the gonad of different genders, with a higher expression in ovary than in testis. In situ hybridization(ISH) results demonstrated Pofigla was specifically expressed in germ cells including oocytes, spermatogonia and spermatocytes. By screening and analyzing two proximal regions(-2966/-2126 and-772/-444) with high promoter activity, we found SOX5, LEF1, FOXP1 and GATA1 may play important roles in the transcriptional regulation of Pofigla. Furthermore, we observed the co-localization between Figla and LEF1 in HEK 293T cells. And the significant up-regulation effect of the canonical Wnt signaling pathway on the expression of Pofigla was found in cultured ovarian cells. This study provided the first evidence that figla not only has an important function in ovary development, but also plays some potential roles in testis development and/or male germ cell differentiation during early testis development in P. olivaceus. The results provide valuable reference in exploring the regulatory network of figla in teleost.

Hypothalamic-Pituitary-Gonadal(HPG)Axis and Transcriptional Regulatory Elements Regulate piwil2 Gene Expression During Gametogenesis and Gonadal Development in Japanese Flounder(Paralichthys olivaceus)

The P-element induced wimpy testis(Piwi)proteins,which are associated with PIWI-interacting RNAs(piRNAs),play important roles in meiosis,germ cell division,and germline maintenance.In this study,we identified and characterized the Paralichthys olivaceus piwil2 gene,a constituent factor of the piRNA pathways involved in the biogenesis of reproductive development.The biological analysis indicated that piwil2,which contains PAZ and PIWI domains,was highly conserved between teleosts and tetrapods.The piwil2 distribution profile in different tissues confirmed a sexually dimorphic expression pattern,with a higher expression level in testis.In situ hybridization demonstrated that piwil2 was expressed in the oogonia and oocytes of the ovaries as well as in the Sertoli cells and spermatocytes of the testes.Gene piwil2 showed a maternally inherited expression pattern during embryonic development,and was highly expressed during the early embryonic development.Different luciferase reporters were constructed to determine the transcriptional regulatory mechanisms of piwil2.The piwil2 core promoter region was located at−360 bp to−60 bp.Furthermore,some representative sex hormones,including human chorionic gonadotropin,17α-methyltestosterone,and estradiol-17βhad distinct regulatory effects on piwil2.In a summery,these results indicate that piwil2,regulated by sex hormones and transcriptional elements,has vital functions in the reproductive cycle and gonadal development.

MiR-430 Can Affect the Mesoderm Formation and Metamorphosis of Paralichthys olivaceus by Targeting lefty Gene

MiR-430 is a non-coding small RNA that participates in early embryonic development by acting on the Nodal signaling pathway.Its functions in Paralichthys olivaceus(P.olivaceus),however,remain unclear.In this study,through biological information technology,we hypothesized lefty was the target gene of miR-430.Lefty is a divergent member of the transforming growth factor-β(TGF-β)superfamily,and is involved in regulating left-right patterning as an antagonist of the Nodal signaling pathway.To study the relationship between miR-430 and lefty gene,miR-430 and fluorescent expression vector were transfected into embryo cells.Our results showed that miR-430 has important effects on early embryonic development.It can inhibit the expressions of lefty and other genes related to Nodal signaling pathway.Additionally,the expression of miR-430 was involved in the metamorphosis of P.olivaceus.This study clarifies the function of miR-430 in P.olivaceus,which will be helpful for further research on the interactions between miR-430 and the Nodal signaling pathway.