Long non-coding RNAs(lncRNAs)are a class of transcripts longer than 200 bp,which have been emerged as essential regulators in numerous biological processes.Black rockfish(Sebastes schlegelii)is an economic fish that w...Long non-coding RNAs(lncRNAs)are a class of transcripts longer than 200 bp,which have been emerged as essential regulators in numerous biological processes.Black rockfish(Sebastes schlegelii)is an economic fish that widely cultured in the coastal areas of China,Japan,and South Korea.With the expansion of aquacultural scale,various pathogens have threatened its industry and reduced its economic values.It has been reported that lncRNA were involved in the immune response and metabolic pathway in teleost,while no study is available on identification and functional analysis of lncRNAs in black rockfish so far.Herein,this study was performed to identify lncRNAs in the intestine of black rockfish after Edwardsiella tarda infection.In our results,a total of 9311 lncRNAs were identified through highthroughput sequencing,and 102 lncRNAs were significantly regulated following challenge,which were predicted to target 3348 mRNAs.Results of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses of the se target genes showed they were function in catalytic activity,hydrolase activity,defense response and peptidase activity,which involved in metabolic pathways and immune related pathways.In addition,47 lncRNAs and 8 differentially expressed mRNAs(DEmRNAs)showed co-expression at two or more infection time points with metabolism and immunity functions.Moreover,real-time quantitative PCR(qRT-PCR)was performed to verify the reliability of sequencing gene expression analysis results.This research laid the foundation for further investigation of the regulatory roles of lncRNAs in the intestinal immune response of black rockfish.展开更多
Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) wit...Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrB-LAMP). In this method, the Mg 2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene (hemolysin-LAMP) for detection of E. tarda. The EsrB-LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples.展开更多
Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In t...Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （logl0n~ as x; n~ is copy number） of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.展开更多
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 m...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.展开更多
Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgent...Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgently.To achieve this purpose,we developed a TaqMan-based real-time PCR assay for detection and quantification of E.tarda.The assay targets the hemolysin activator HlyB domain protein of E.tarda.Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction.A standard curve was generated from the threshold cycle values(y) against log10(E.tarda genomic DNA concentration) as x.The intra-and inter-assay coefficient of variation(CV) values were less than 2.06% and 1.05% respectively,indicating that the assay had good reproducibility.This method is highly specific to E.tarda strains,as it shows no cross-reactivity to Edwardsiella ictaluri,a member of the same genus,or to nine other fish-pathogenic bacteria species belonging to three other genera.This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E.tarda in clinical samples.展开更多
Over the past five years, an epidemic disease broke out in Pelteobagrus fulvidraco ponds of Zhejiang Province. The diseased fish mainly exhibits head splitting and surface bleeding; dissection results reveal hepatonep...Over the past five years, an epidemic disease broke out in Pelteobagrus fulvidraco ponds of Zhejiang Province. The diseased fish mainly exhibits head splitting and surface bleeding; dissection results reveal hepatonephromegaly and abdominal bleeding. A bacterial strain HSY201301 was isolated from the liver tissue of P. fulvidraco with typical symptoms. Artificial infection experiment confirmed that the isolated strain had a strong virulence to healthy P. fidvidraco, leading to similar symptoms to naturally infected P. fulvidraco. The isolated strain was identified as an Edwardsiella tarda strain according to conventional morphological, physiological, biochemical characteristics and 16S rRNA gene sequence. Results of drug susceptibility test indicated that the isolated strain was sensitive to cipro- floxacin, doxitard, penicillin, doxycycline, and rocephin. This study laid solid foundation for effective prevention and control of E. tarda.展开更多
目前,细菌性疾病是中华鳖养殖中的常见疾病。实验采用常规方法从呈白底板症状的中华鳖(Pelodiscus sinensis)肝脏、脾脏、肾脏、胃积液等中分离纯化病原菌,经形态观察、生理生化鉴定和16S r RNA序列分析确定其种属,通过回归感染实验判...目前,细菌性疾病是中华鳖养殖中的常见疾病。实验采用常规方法从呈白底板症状的中华鳖(Pelodiscus sinensis)肝脏、脾脏、肾脏、胃积液等中分离纯化病原菌,经形态观察、生理生化鉴定和16S r RNA序列分析确定其种属,通过回归感染实验判断分离菌株致病性,并以纸片扩散法测定其药物敏感性。结果显示:自病鳖中分离可得到一种优势菌落,其表面湿润光滑、微隆起、半透明,呈灰黄色;基于VITEK 2的64个生化反应的分析提示,分离菌理化特性与迟缓爱德华氏菌(Edwardsiella tarda)基本一致;16S rRNA序列分析显示,其与迟缓爱德华氏菌同源性达98%以上。腹腔注射回归感染实验引起稚鳖死亡,且细菌分离能再次得到相同分离株,表明分离到的迟缓爱德华氏菌具有致病性。药敏试验表明该菌对庆大霉素、阿米卡星、新霉素、头孢哌酮、氨曲南等5种抗生素较为敏感。本研究可为中华鳖病害的正确诊断和科学防治提供参考。展开更多
A study was carried out to investigate antibiotic resistance patterns and plasmid profiling of Edwardsiella tarda isolated from farmed-cultured Heterobranchus longifilis in Lagos State, Southwest of Nigeria. A total o...A study was carried out to investigate antibiotic resistance patterns and plasmid profiling of Edwardsiella tarda isolated from farmed-cultured Heterobranchus longifilis in Lagos State, Southwest of Nigeria. A total of 44 Edwardsiella isolates were recovered from 80 fish samples collected from the 10 fish farms using selective random stratification. It was observed that Edwardsiella tarda isolates were 100% resistant to Amoxicillin, Chloranphenicol, Levofloxacin, Streptomycin and 90% resistant to Nalidixic Acid respectively. All the isolates were 100% susceptible to Spectinomycin and Ciprofloxacin, while Ofloxacin, Gentamycin, and Pefloxacin vary in their level of susceptibility with 90%, 80% and 70% sensitivity respectively. Conversely, 8 out of 10 fish farm locations studied were observed to have antibiotic-resistant strains, and 5 out of 8 drug-resistant strains were found to carry plasmid and the sizes of the plasmid ranges between 20.027 kb to 23.130 kb. The plasmid after treatment with mitomycin C and ethidium bromide were lost during the process of plasmid curing confirming that the multiple drug resistant exhibited by the isolates was plasmid mediated. There are fewer studies on antibiotic resistance in Edwardsiella tarda from aquaculture enterprises and this study provides further support to the view that there is a potential risk of transfer of resistant bacteria and their genes to human pathogen through the food chain. Although, in Nigeria there is no antibiotic product registered for aquaculture usage, yet fish farmers use them off-label for bacterial diseases prevention.展开更多
The E. tarda bacterial culture isolated from infected fish, was confirmed by morphological and biochemical characterization. GAPDH gene of 996 bp was amplified from bacterial genomic DNA. GAPDH gene was cloned in pTZ5...The E. tarda bacterial culture isolated from infected fish, was confirmed by morphological and biochemical characterization. GAPDH gene of 996 bp was amplified from bacterial genomic DNA. GAPDH gene was cloned in pTZ57R/T cloning vector. The positive clone was sequenced. The sequencing result showed very homology with published sequence from pathogenic E. tarda. The sequence (nucleotide and amino acid) divergence values were very low between E. tarda isolates from India (KF142190) and China (HQ697337-38) (Figure 4 and Figure 5). However, the divergence value was high when compared with E. tarda isolates from Japan (AB198939) and this value was higher than the inter-specific divergence value (E. tarda-E. ictaluri, E. tarda-E. coli & E. tarda-Klebsiella species).展开更多
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 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.展开更多
Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rock...Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.展开更多
基金Supported by the Young Experts of Taishan Scholars(No.tsqn201909130)the Science and Technology Support Plan for Youth Innovation of Colleges and Universities in Shandong Province(No.2019KJF003)+1 种基金the“First Class Fishery Discipline”Program in Shandong Province,a special talent program“One Matter One Decision(Yi Shi Yi Yi)”Program in Shandong Province,Chinathe Breeding Plan of Shandong Provincial Qingchuang Research Team(2019)。
文摘Long non-coding RNAs(lncRNAs)are a class of transcripts longer than 200 bp,which have been emerged as essential regulators in numerous biological processes.Black rockfish(Sebastes schlegelii)is an economic fish that widely cultured in the coastal areas of China,Japan,and South Korea.With the expansion of aquacultural scale,various pathogens have threatened its industry and reduced its economic values.It has been reported that lncRNA were involved in the immune response and metabolic pathway in teleost,while no study is available on identification and functional analysis of lncRNAs in black rockfish so far.Herein,this study was performed to identify lncRNAs in the intestine of black rockfish after Edwardsiella tarda infection.In our results,a total of 9311 lncRNAs were identified through highthroughput sequencing,and 102 lncRNAs were significantly regulated following challenge,which were predicted to target 3348 mRNAs.Results of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses of the se target genes showed they were function in catalytic activity,hydrolase activity,defense response and peptidase activity,which involved in metabolic pathways and immune related pathways.In addition,47 lncRNAs and 8 differentially expressed mRNAs(DEmRNAs)showed co-expression at two or more infection time points with metabolism and immunity functions.Moreover,real-time quantitative PCR(qRT-PCR)was performed to verify the reliability of sequencing gene expression analysis results.This research laid the foundation for further investigation of the regulatory roles of lncRNAs in the intestinal immune response of black rockfish.
基金Supported by the Special Fund for Agro-Scientific Research in the Public Interest (No. 201103034)the Earmarked Fund for Modern Agroindustry Technology Research System (No. nycytx-46)
文摘Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrB-LAMP). In this method, the Mg 2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene (hemolysin-LAMP) for detection of E. tarda. The EsrB-LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples.
基金The Special Fund for Agro-scientific Research in the Public Interest under contract No.201103034Construction Special Fund of Modern Agriculture and Industrial Technology Research System under contract No.CARS-47
文摘Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （logl0n~ as x; n~ is copy number） of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.
基金supported by the Applied Basic Research Programs of the Science and Technology Commission Foundation of Tianjin, China (Nos. 19JCZDJC34300, 14JCZDJC34200 and 18JCYBJC96100)
文摘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.
基金Supported by the Special Fund for Agro-scientific Research in the Public Interest(No.201103034)the Construction Special Fund of Modern Agriculture and Industrial Technology Research System(No.CARS-47)
文摘Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgently.To achieve this purpose,we developed a TaqMan-based real-time PCR assay for detection and quantification of E.tarda.The assay targets the hemolysin activator HlyB domain protein of E.tarda.Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction.A standard curve was generated from the threshold cycle values(y) against log10(E.tarda genomic DNA concentration) as x.The intra-and inter-assay coefficient of variation(CV) values were less than 2.06% and 1.05% respectively,indicating that the assay had good reproducibility.This method is highly specific to E.tarda strains,as it shows no cross-reactivity to Edwardsiella ictaluri,a member of the same genus,or to nine other fish-pathogenic bacteria species belonging to three other genera.This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E.tarda in clinical samples.
文摘Over the past five years, an epidemic disease broke out in Pelteobagrus fulvidraco ponds of Zhejiang Province. The diseased fish mainly exhibits head splitting and surface bleeding; dissection results reveal hepatonephromegaly and abdominal bleeding. A bacterial strain HSY201301 was isolated from the liver tissue of P. fulvidraco with typical symptoms. Artificial infection experiment confirmed that the isolated strain had a strong virulence to healthy P. fidvidraco, leading to similar symptoms to naturally infected P. fulvidraco. The isolated strain was identified as an Edwardsiella tarda strain according to conventional morphological, physiological, biochemical characteristics and 16S rRNA gene sequence. Results of drug susceptibility test indicated that the isolated strain was sensitive to cipro- floxacin, doxitard, penicillin, doxycycline, and rocephin. This study laid solid foundation for effective prevention and control of E. tarda.
文摘A study was carried out to investigate antibiotic resistance patterns and plasmid profiling of Edwardsiella tarda isolated from farmed-cultured Heterobranchus longifilis in Lagos State, Southwest of Nigeria. A total of 44 Edwardsiella isolates were recovered from 80 fish samples collected from the 10 fish farms using selective random stratification. It was observed that Edwardsiella tarda isolates were 100% resistant to Amoxicillin, Chloranphenicol, Levofloxacin, Streptomycin and 90% resistant to Nalidixic Acid respectively. All the isolates were 100% susceptible to Spectinomycin and Ciprofloxacin, while Ofloxacin, Gentamycin, and Pefloxacin vary in their level of susceptibility with 90%, 80% and 70% sensitivity respectively. Conversely, 8 out of 10 fish farm locations studied were observed to have antibiotic-resistant strains, and 5 out of 8 drug-resistant strains were found to carry plasmid and the sizes of the plasmid ranges between 20.027 kb to 23.130 kb. The plasmid after treatment with mitomycin C and ethidium bromide were lost during the process of plasmid curing confirming that the multiple drug resistant exhibited by the isolates was plasmid mediated. There are fewer studies on antibiotic resistance in Edwardsiella tarda from aquaculture enterprises and this study provides further support to the view that there is a potential risk of transfer of resistant bacteria and their genes to human pathogen through the food chain. Although, in Nigeria there is no antibiotic product registered for aquaculture usage, yet fish farmers use them off-label for bacterial diseases prevention.
文摘The E. tarda bacterial culture isolated from infected fish, was confirmed by morphological and biochemical characterization. GAPDH gene of 996 bp was amplified from bacterial genomic DNA. GAPDH gene was cloned in pTZ57R/T cloning vector. The positive clone was sequenced. The sequencing result showed very homology with published sequence from pathogenic E. tarda. The sequence (nucleotide and amino acid) divergence values were very low between E. tarda isolates from India (KF142190) and China (HQ697337-38) (Figure 4 and Figure 5). However, the divergence value was high when compared with E. tarda isolates from Japan (AB198939) and this value was higher than the inter-specific divergence value (E. tarda-E. ictaluri, E. tarda-E. coli & E. tarda-Klebsiella species).
基金Supported by the National Natural Science Foundation of China(No.31461163005)the Taishan Scholar Project of Shandong Province
文摘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.
基金supported by the Natural Science Foundation of Shandong Province(No.ZR2020QC214)the Young Experts of Taishan Scholars(No.tsqn201909130)+3 种基金the Science and Technology Support Plan for Youth Innovation of Colleges and Universities in Shandong Province(No.2019KJF003)the‘First Class Fishery Discipline’Programme in Shandong Provincea special talent programme‘One Thing One Decision(YishiYiyi)’Programme in Shandong Province,Chinathe Breeding Plan of Shandong Provincial Qingchuang Research Team(2019)。
文摘Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.
