VIGS(Virus-induced gene silencing),a method for posttranscriptional gene silencing,is an effective technique for investigating the activities of genes in plants.Since there is no report for available VIGS system in St...VIGS(Virus-induced gene silencing),a method for posttranscriptional gene silencing,is an effective technique for investigating the activities of genes in plants.Since there is no report for available VIGS system in Styrax japonicus,the application of a VIGS approach that results in a gene knockdown to study gene function is limited.In this study,we compared the characteristics that could affect the viability of VIGS in S.japonicus,including the acetosyringone(AS)concentration,the Agrobacterium’s optical density and the inoculation method.The stable reference genes of S.japonicus were selected to validate the gene’s knockdown by quantitative PCR.As a result,we successfully constructed 2 VIGS systems based on TRV virus:vacuum with AS concentration of 200μmol·L^(-1)and OD600of 0.5,and friction-osmosis with AS concentration of 200μmol·L^(-1)and OD600of 1.0,which silencing efficiency was 83.33%and 74.19%,respectively.The successfully applied VIGS method provides a rapid and effective reverse gene functional analysis approach in S.japonicus to identify unknown gene functions.展开更多
Information on survival and growth during the early life stage is essential to understand the mechanism of interannual variations in fish recruitment.Chub mackerel Scomber japonicus is a commercially important pelagic...Information on survival and growth during the early life stage is essential to understand the mechanism of interannual variations in fish recruitment.Chub mackerel Scomber japonicus is a commercially important pelagic fish widely distributed in the northwestern Pacific.Its catch showed large fluctuations with changes in distribution and migration under climate change and strong fishing.We determined the hatch dates and growth rates of young-of-the-year of chub mackerel through otolith microstructure using samples collected in the Oyashio water in autumn 2018.Results show that the ages of young chub mackerel ranged between 120 and 180 d,and the estimated hatch date lasted from midJanuary to late May with a peak from mid-March to mid-April.Average otolith daily increment width during the early life stages(from hatching to 25 d)showed an increasing trend.Chub mackerel grows slowly in the first 10 d,and then grows faster during the 10thto 25thd.Three groups with dissimilar growth histories and migration routes were identified using unsupervised random forest clustering analysis,but all eventually converge on the same nursery ground.The faster growth of young-of-the-year chub mackerel leads to better recruitment due to the hypothesis of growth-dependent mortality.Most chub mackerels hatched in March and April,the spawning period is longer and earlier,which could lead to strong year classes.These findings on population composition and life history traits of young-of-the-year of chub mackerel provide valuable information on its recruitment processes during the period of stock recovery.展开更多
The sea cucumber Apostichopus japonicus plays important roles in marine benthic ecosystem as environmental cleaners,and it is the important aquaculture species in China.High water temperature poses critical threat for...The sea cucumber Apostichopus japonicus plays important roles in marine benthic ecosystem as environmental cleaners,and it is the important aquaculture species in China.High water temperature poses critical threat for the survival of A.japonicus,which has resulted in extensive death in summer.To explore the genes expression profiles under different levels of heat stress,the high-throughput RNA-seq was applied in this study.Our results revealed a total of 1371,1225 and 1408 differentially expressed genes(DEGs)in 26℃for 6 h,26℃for 48 h and 30℃for 6 h respectively in comparison with Control group.The pathway analysis suggested“Protein processing in endoplasmic reticulum(ER)”was significantly enriched in all these heat stress(HS)treatment groups.The expression results of key DEGs in this pathway(Hsp70,Derlin,NEF,PDI,GPR94 and ERP57)by qRT-PCR was in accordance with the RNA-seq data.The subcluster analysis of DEGs revealed that a variety of heat shock proteins(HSPs)and calcium ion binding proteins had an obvious up-regulated expression in 26℃for 6 h,comparatively low expression in 26℃for 48 h,and the highest expression in 30℃for 6 h.The other DEGs subcluster,consisting of critical components of extracellular matrix(ECM)and a subset of peptidases and proteases,showed significantly rising tendency in 30℃for 6 h.Additionally,the expression of matrix metalloproteases(MMP1,MMP16 and MMP19)was prominently affected by HS,and peaked in 30℃for 6 h.This study provides a series of candidate genes for further study about heat shock response in A.japonicus,especially genes associated with protein processing in ER and regulation of ECM,which also offers new insights into cellular homeostasis under stressful conditions in marine invertebrates.展开更多
Sea cucumber(Apostichopus japonicus)is an excellent model for investigating effects of bottom-dwellers on carbon mig-ration and transformation.However,the molecular mechanism of respiratory metabolism process variatio...Sea cucumber(Apostichopus japonicus)is an excellent model for investigating effects of bottom-dwellers on carbon mig-ration and transformation.However,the molecular mechanism of respiratory metabolism process variation caused by feeding rations is poorly understood.In this study,treatment groups set as 1%(about 0.63g),3%,and 7%of total body weight(named F1,F3 and F7 groups respectively).The potential molecular mechanisms behind the functions of respiratory tree and body wall were investigated by RNA-Seq.A total of 52411 expressed genes were identified from 89342 expressed transcripts.The results showed 759,254 and 334 genes were up-regulated,and 334,445 and 992 genes were down-regulated in respiratory tree of F1 vs.F3,F1 vs.F7 and F3 vs.F7,respectively.Meanwhile,2070,1601 and 896 genes were up-regulated,and 1303,1337 and 1144 genes were down-regulated in body wall between F1 vs.F3,F1 vs.F7 and F3 vs.F7,respectively.Differentially expressed genes were enriched in salivary secretion and ECM-receptor interaction pathways in respiratory tree,and in various types of N-glycan biosynthesis,ribosome and sphingolipid metabolism pathways in body wall.These results suggested respiratory tree and body wall were involved in activation of respiratory metabolisms in response to different feeding rations.Our research provided valuable knowledge for physiological differences in res-piratory metabolism.展开更多
Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes,leading to the production of excessive reactive oxygen species(ROS)and irreversible apopt...Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes,leading to the production of excessive reactive oxygen species(ROS)and irreversible apoptotic cell death.Emerging evidence suggests that mitochondrial autophagy(mitophagy)is the most effective method for eliminating damaged mitochondria and ROS,with choline dehydrogenase(CHDH)identified as a novel mitophagy receptor that can recognize non-ubiquitin damage signals and microtubule-associated protein 1 light chain 3(LC3)in vertebrates.However,the functional role of CHDH in invertebrates is largely unknown.In this study,we observed a significant increase in the mRNA and protein expression levels of A.japonicus CHDH(AjCHDH)in response to V.splendidus infection and lipopolysaccharide(LPS)challenge,consistent with changes in mitophagy under the same conditions.Notably,AjCHDH was localized to the mitochondria rather than the cytosol following V.splendidus infection.Moreover,AjCHDH knockdown using si RNA transfection significantly reduced mitophagy levels,as observed through transmission electron microscopy and confocal microscopy.Further investigation into the molecular mechanisms underlying CHDH-regulated mitophagy showed that AjCHDH lacked an LC3-interacting region(LIR)for direct binding to LC3 but possessed a FB1 structural domain that binds to SQSTM1.The interaction between AjCHDH and SQSTM1 was further confirmed by immunoprecipitation analysis.Furthermore,laser confocal microscopy indicated that SQSTM1 and LC3 were recruited by AjCHDH in coelomocytes and HEK293T cells.In contrast,AjCHDH interference hindered SQSTM1 and LC3 recruitment to the mitochondria,a critical step in damaged mitochondrial degradation.Thus,AjCHDH interference led to a significant increase in both mitochondrial and intracellular ROS,followed by increased apoptosis and decreased coelomocyte survival.Collectively,these findings indicate that AjCHDH-mediated mitophagy plays a crucial role in coelomocyte survival in A.japonicus following V.splendidus infection.展开更多
基金supported by the Subject of Key R&D Plan of Shandong Province(Major Scientific and Technological Innovation Project)“Mining and Accurate Identification of Forest Tree Germplasm Resources”(Grant Nos.2021LZGC02303)Science&Technology Specific Projects in Agricultural High-tech Industrial Demonstration Area of the Yellow River Delta(Grant No.022SZX16)。
文摘VIGS(Virus-induced gene silencing),a method for posttranscriptional gene silencing,is an effective technique for investigating the activities of genes in plants.Since there is no report for available VIGS system in Styrax japonicus,the application of a VIGS approach that results in a gene knockdown to study gene function is limited.In this study,we compared the characteristics that could affect the viability of VIGS in S.japonicus,including the acetosyringone(AS)concentration,the Agrobacterium’s optical density and the inoculation method.The stable reference genes of S.japonicus were selected to validate the gene’s knockdown by quantitative PCR.As a result,we successfully constructed 2 VIGS systems based on TRV virus:vacuum with AS concentration of 200μmol·L^(-1)and OD600of 0.5,and friction-osmosis with AS concentration of 200μmol·L^(-1)and OD600of 1.0,which silencing efficiency was 83.33%and 74.19%,respectively.The successfully applied VIGS method provides a rapid and effective reverse gene functional analysis approach in S.japonicus to identify unknown gene functions.
基金Supported by the National Natural Science Foundation of China(No.41930534)the Third Institute of Oceanography through the National Program on Global Change and Air-Sea Interaction(No.GASI-02-PACYDaut)。
文摘Information on survival and growth during the early life stage is essential to understand the mechanism of interannual variations in fish recruitment.Chub mackerel Scomber japonicus is a commercially important pelagic fish widely distributed in the northwestern Pacific.Its catch showed large fluctuations with changes in distribution and migration under climate change and strong fishing.We determined the hatch dates and growth rates of young-of-the-year of chub mackerel through otolith microstructure using samples collected in the Oyashio water in autumn 2018.Results show that the ages of young chub mackerel ranged between 120 and 180 d,and the estimated hatch date lasted from midJanuary to late May with a peak from mid-March to mid-April.Average otolith daily increment width during the early life stages(from hatching to 25 d)showed an increasing trend.Chub mackerel grows slowly in the first 10 d,and then grows faster during the 10thto 25thd.Three groups with dissimilar growth histories and migration routes were identified using unsupervised random forest clustering analysis,but all eventually converge on the same nursery ground.The faster growth of young-of-the-year chub mackerel leads to better recruitment due to the hypothesis of growth-dependent mortality.Most chub mackerels hatched in March and April,the spawning period is longer and earlier,which could lead to strong year classes.These findings on population composition and life history traits of young-of-the-year of chub mackerel provide valuable information on its recruitment processes during the period of stock recovery.
基金The National Natural Science Foundation of China under contract Nos 42276143 and 31902360the Shandong Provincial Natural Science Foundation under contract Nos ZR2022MC050 and ZR2022QD003the“First Class Fishery Discipline”Programme[(2020)3]in Shandong Province。
文摘The sea cucumber Apostichopus japonicus plays important roles in marine benthic ecosystem as environmental cleaners,and it is the important aquaculture species in China.High water temperature poses critical threat for the survival of A.japonicus,which has resulted in extensive death in summer.To explore the genes expression profiles under different levels of heat stress,the high-throughput RNA-seq was applied in this study.Our results revealed a total of 1371,1225 and 1408 differentially expressed genes(DEGs)in 26℃for 6 h,26℃for 48 h and 30℃for 6 h respectively in comparison with Control group.The pathway analysis suggested“Protein processing in endoplasmic reticulum(ER)”was significantly enriched in all these heat stress(HS)treatment groups.The expression results of key DEGs in this pathway(Hsp70,Derlin,NEF,PDI,GPR94 and ERP57)by qRT-PCR was in accordance with the RNA-seq data.The subcluster analysis of DEGs revealed that a variety of heat shock proteins(HSPs)and calcium ion binding proteins had an obvious up-regulated expression in 26℃for 6 h,comparatively low expression in 26℃for 48 h,and the highest expression in 30℃for 6 h.The other DEGs subcluster,consisting of critical components of extracellular matrix(ECM)and a subset of peptidases and proteases,showed significantly rising tendency in 30℃for 6 h.Additionally,the expression of matrix metalloproteases(MMP1,MMP16 and MMP19)was prominently affected by HS,and peaked in 30℃for 6 h.This study provides a series of candidate genes for further study about heat shock response in A.japonicus,especially genes associated with protein processing in ER and regulation of ECM,which also offers new insights into cellular homeostasis under stressful conditions in marine invertebrates.
基金supported by the National Natural Science Foundation of China(No.31672657).
文摘Sea cucumber(Apostichopus japonicus)is an excellent model for investigating effects of bottom-dwellers on carbon mig-ration and transformation.However,the molecular mechanism of respiratory metabolism process variation caused by feeding rations is poorly understood.In this study,treatment groups set as 1%(about 0.63g),3%,and 7%of total body weight(named F1,F3 and F7 groups respectively).The potential molecular mechanisms behind the functions of respiratory tree and body wall were investigated by RNA-Seq.A total of 52411 expressed genes were identified from 89342 expressed transcripts.The results showed 759,254 and 334 genes were up-regulated,and 334,445 and 992 genes were down-regulated in respiratory tree of F1 vs.F3,F1 vs.F7 and F3 vs.F7,respectively.Meanwhile,2070,1601 and 896 genes were up-regulated,and 1303,1337 and 1144 genes were down-regulated in body wall between F1 vs.F3,F1 vs.F7 and F3 vs.F7,respectively.Differentially expressed genes were enriched in salivary secretion and ECM-receptor interaction pathways in respiratory tree,and in various types of N-glycan biosynthesis,ribosome and sphingolipid metabolism pathways in body wall.These results suggested respiratory tree and body wall were involved in activation of respiratory metabolisms in response to different feeding rations.Our research provided valuable knowledge for physiological differences in res-piratory metabolism.
基金supported by the National Natural Science Foundation of China (32102825)Natural Science Foundation of Zhejiang Province (LQ22C190003)。
文摘Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes,leading to the production of excessive reactive oxygen species(ROS)and irreversible apoptotic cell death.Emerging evidence suggests that mitochondrial autophagy(mitophagy)is the most effective method for eliminating damaged mitochondria and ROS,with choline dehydrogenase(CHDH)identified as a novel mitophagy receptor that can recognize non-ubiquitin damage signals and microtubule-associated protein 1 light chain 3(LC3)in vertebrates.However,the functional role of CHDH in invertebrates is largely unknown.In this study,we observed a significant increase in the mRNA and protein expression levels of A.japonicus CHDH(AjCHDH)in response to V.splendidus infection and lipopolysaccharide(LPS)challenge,consistent with changes in mitophagy under the same conditions.Notably,AjCHDH was localized to the mitochondria rather than the cytosol following V.splendidus infection.Moreover,AjCHDH knockdown using si RNA transfection significantly reduced mitophagy levels,as observed through transmission electron microscopy and confocal microscopy.Further investigation into the molecular mechanisms underlying CHDH-regulated mitophagy showed that AjCHDH lacked an LC3-interacting region(LIR)for direct binding to LC3 but possessed a FB1 structural domain that binds to SQSTM1.The interaction between AjCHDH and SQSTM1 was further confirmed by immunoprecipitation analysis.Furthermore,laser confocal microscopy indicated that SQSTM1 and LC3 were recruited by AjCHDH in coelomocytes and HEK293T cells.In contrast,AjCHDH interference hindered SQSTM1 and LC3 recruitment to the mitochondria,a critical step in damaged mitochondrial degradation.Thus,AjCHDH interference led to a significant increase in both mitochondrial and intracellular ROS,followed by increased apoptosis and decreased coelomocyte survival.Collectively,these findings indicate that AjCHDH-mediated mitophagy plays a crucial role in coelomocyte survival in A.japonicus following V.splendidus infection.
