The yellow-colored line of pearl oyster Pinctada fucata martensii displays a yellow prismatic layer and a white nacreous layer that can be used as an ideal model for research on shell color formation.Micro-Raman spect...The yellow-colored line of pearl oyster Pinctada fucata martensii displays a yellow prismatic layer and a white nacreous layer that can be used as an ideal model for research on shell color formation.Micro-Raman spectroscopy and transcriptome analyses were performed to explore the potential molecular mechanism underlying the phenotype differentiation.The micro-Raman spectroscopy results indicate that the prismatic layer exhibits distinct characteristic peaks of carotenoids,while these peaks are not prominent in the nacreous layer.In the transcriptome comparison of the central zone of mantle and mantle edge tissue,which function in nacreous and prismatic layer formation,respectively,935 significantly differentially expressed genes(DEGs)were identified,with 385 genes upregulated and 227 genes downregulated(|log_(2)(Fold change)|>1 and false discovery rate<0.05)in the mantle edge tissue.Among these genes,some were associated with melanoma/melanogenesis,such as tyrosinase,zinc metalloprotease,glutathione S-transferase,and ATP-binding cassette sub-family;some were associated with the carotenoid-related pathway,including scavenger receptors,cytochrome P450 and lipoprotein receptor.Genes associated with porphyrin metabolism,including porphobilinogen deaminase,and copper/zinc superoxide dismutase,and genes associated with shell matrix protein,including amorphous calcium carbonate binding protein,shematrin,PIF,and collagen,also exhibited significantly different expressions.It is speculated that the different colours between prismatic layer and nacreous layer in the yellow-colored line of P.f.martensii might be resulted from melanin,carotenoids and porphyrin metabolism,while genes related to shell structure and biomineralization might also affect coloration.Our results provide new insights to understand the mechanism of shell color formation in mollusca.展开更多
Inhibitory Smads(I-Smads),which belong to the Smad family and inhibit bone morphogenic protein 2(BMP2)signaling by a variety of mechanisms,can suppress innate immunity responses in vertebrates.However,there are no rep...Inhibitory Smads(I-Smads),which belong to the Smad family and inhibit bone morphogenic protein 2(BMP2)signaling by a variety of mechanisms,can suppress innate immunity responses in vertebrates.However,there are no reports for the role of Smad6 in immunity in mollusks.In this study,we showed that Smad6 of the pearl oyster Pinctada fucata martensii was located in the Smad6 cluster of the phylogenetic tree;mRNA expression of Smad6 and Smad3 was up-regulated after lipopolysaccharide and polyinosinic:polycytidylic challenge;and transcript levels of Smad6 and Smad3 showed opposite patterns during wound healing.Under salinity stress,water inflow and outflow in the gills appear to be regulated by BMP2-Smads signals,and BMP2-Smads signaling may be closely related to the immune response.Our results indicate that Smad6 is involved in immunity,that it plays a positive role in the response to immune challenge and an inhibitory role during wound healing,and that Smad6 and Smad3 may work against each other.展开更多
The pearl oyster Pinctada fucata martensii is an economically valuable shellfish that is cultured for seawater pearl pro-duction,which mainly depends on oyster growth.However,the growth mechanisms of the pearl oyster ...The pearl oyster Pinctada fucata martensii is an economically valuable shellfish that is cultured for seawater pearl pro-duction,which mainly depends on oyster growth.However,the growth mechanisms of the pearl oyster are still poorly understood.In this study,oysters were grouped with relative growth rate,including fast-growing(FG)group and slow-growing(SG)group.Oxford Nanopore Technologies(ONT)long-read sequencing was applied to investigate the molecular mechanisms involved in the growth of this species.Five alternative splicing(AS)types were analyzed in both FG and SG groups,which include alternative 3’splice site,alternative 5’splice site,exon skipping,intron retention,and mutually exclusive exon.Transcriptome analysis showed that four of five different AS events(excluding mutually exclusive exons)occurred more frequently in FG than in SG oysters,and the five main AS types exhibited different characteristics.The AS events that were detected may be involved in growth,and the difference in ex-pression of AS events between FG and SG oysters may be involved in the mechanism underlying the difference in growth.Fifty dif-ferentially expressed genes(DEGs)were identified between the FG and SG oysters.The results showed that 40 genes were signifi-cantly up-regulated in FG oysters,while 10 genes were significantly down-regulated in SG oyster.Several genes related to nutrient metabolism,shell formation,and immunity were more highly expressed in FG oysters than in SG oysters.In summary,FG oysters exhibited higher metabolic and biomineralization activities and had a more powerful immune system than SG oysters.These results provide insight into the growth of P.f.martensii that can be used to improve breeding programs.展开更多
Differentially expressed genes(DEGs)between individuals with high(HC)and low(LC)total carotenoid content(TCC)were sampled from a selected line of Pinctada fucata martensii with black shell in the prismatic layer.The e...Differentially expressed genes(DEGs)between individuals with high(HC)and low(LC)total carotenoid content(TCC)were sampled from a selected line of Pinctada fucata martensii with black shell in the prismatic layer.The expression levels of candidate genes were verified by qRT-PCR.Targeted resequencing was used to detect SNPs in a candidate gene,PmSR-BI.The association of TCC with SNPs in PmSR-BI was determined.Results showed that a total of 1025 DEGs were identified between HC and LC.The expression levels of the candidate gene PmSR-BI in HC were higher than those in LC.Seven SNPs in the exon and eight SNPs in the 5′regulatory regions of PmSR-BI were found.Association analysis showed that one SNP in the exon and two SNPs in the 5′regulatory regions of PmSR-BI were significantly associated with the TCC(P<0.05).All SNPs of PmSR-BI were divided into four blocks.CC haplotype in Block 1 and AG haplotype in Block 3 were significantly higher than other haplotypes.These results help elucidate the mechanism underlying carotenoid metabolism and develop marker-assisted breeding design in the species.展开更多
[目的]开发马氏珠母贝(Pinctada fucata)基因组资源,挖掘功能基因。[方法]以马氏珠母贝血细胞为试材,利用单分子实时技术(single-molecule real time,SMRT)进行全长转录组测序,并对所获得unigenes进行功能注释和基因结构分析。[结果]共...[目的]开发马氏珠母贝(Pinctada fucata)基因组资源,挖掘功能基因。[方法]以马氏珠母贝血细胞为试材,利用单分子实时技术(single-molecule real time,SMRT)进行全长转录组测序,并对所获得unigenes进行功能注释和基因结构分析。[结果]共获得277064条全长非嵌合序列和82381个基因。经过比对nr、SwissProt、KEGG和KOG数据库进行注释和功能分类后,共得到59621个注释基因。同时,在8493条基因中共发现11219个SSR位点,其中以二核苷酸重复基元类型最高(50.9%)。生物信息学分析鉴定得到20013个lncRNA、2004个转录因子、10522个可变剪切分析位点。[结论]使用SMRT技术能够深入挖掘马氏珠母贝全长转录组数据,为进一步探讨马氏珠母贝功能基因的挖掘、免疫响应及遗传机制的研究提供可靠的基因组资源。展开更多
The mechanisms of sex determination and sex differentiation in the pearl oyster P inctada fucata are currently poorly understood. We therefore investigated the roles of orthologs of the D mrt gene family, key players ...The mechanisms of sex determination and sex differentiation in the pearl oyster P inctada fucata are currently poorly understood. We therefore investigated the roles of orthologs of the D mrt gene family, key players in male gonad differentiation in mammals, in P. fucata sex diff erentiation and sexual development. Pf-Dmrt4 exhibits features typical of the D mrt family, and displays significant homologies to the DMRT4 cluster. Pf-Dmrt4 mRNA expression in the gonads during a gametogenic cycle, measured by quantitative polymerase chain reaction, was maximal in mature individuals. P f-Dmrt4 expression, demonstrated by in situ hybridization, was localized in the spermatozoa, spermatids, oocytes and vitellogenic oocytes. Knockdown of Pf-Dmrt4 with double-stranded RNA resulted in decreased mRNA expression levels. And Pf-Dmrt4-dsRNA-injected groups showed spawning-stage male gonads, with ruptured follicles and released spermatozoa. Our results enhance the understanding of sex determination and differentiation in P. fucata and suggest that Pf-Dmrt4 could be involved in male gonadal development, and maintenance of male gonadal function.展开更多
Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that binds Ca2+ and is a thin filament-associated regulator of vertebrate striated muscle contraction. The function of troponin I (TnI) ...Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that binds Ca2+ and is a thin filament-associated regulator of vertebrate striated muscle contraction. The function of troponin I (TnI) in vertebrates has been extensively characterized, but its role in molluscan muscles has not yet been elucidated. Our previous work suggested that the troponin C subunit has a role in adductor phasic muscle but not in catch muscle. Here, we investigated the molecular characteristics of TnI from the bivalve Japanese pearl oyster, Pinctada fucata to aid the elucidation of the function of molluscan muscle troponin. We determined the primary structure of the full-length TnI protein from the P. fucata adductor muscle (Pifuc-TnI) and found that it is composed of 286 amino acid residues with a predicted molecular weight of 33,737. Motif structure predictions and multiple sequence alignments revealed that Pifuc-TnI has a 138 residue extension at its N-terminus compared with rabbit TnI. This is analogous to characterized TnIs from other mollusks. However, unlike scallop TnI, Pifuc-TnI is predicted to contain two cAMP-dependent protein kinase phosphorylation sites, at residues 39 - 45 (RRGTEDD) and 145 - 151 (KKKSKRK). Phylogenetic analysis indicated that Pifuc-TnI and molluscan TnIs were grouped into the same clade. Pifuc-TnI gene structure predictions using Splign alignment of our obtained cDNA and genome sequences indicated that Pifuc-TnI consists of fifteen exons, with the start and stop codons located in exon 2 and exon 11, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TnI gene is predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, and is not expressed in the gill, mantle or foot. These findings suggest that TnI, as a component of the troponin complex, plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction.展开更多
Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has be...Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has been obscure in molluscan muscles. Our previous work indicated that the TnC and TnI subunits work in adductor phasic muscle, but not in catch muscle. Here, we have characterized TnT from the Japanese bivalve pearl oyster Pinctada fucata to start to explain the function of Tn in molluscan muscle contraction. We determined the primary structure of the full-length TnT protein from the P. fucata adductor muscle (Pifuc-TnT), and found that it is composed of 316 amino acid residues with a predicted molecular mass of 37.4 kDa. Multiple sequence alignment showed that Pifuc-TnT has an extension of >60 residues at the C-terminus that are not present in vertebrate TnTs, including known TnTs from other mollusks. Pifuc-TnT gene structure predictions using Splign alignment of the cDNA generated in this study and genome sequences indicated that Pifuc-TnT consists of 13 exons. Start and stop codons are located in exons 2 and 12, respectively. Quantitative real-time PCR revealed that the Pifuc-TnT gene was predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, slightly in gill, and not at all in mantle and foot. These findings suggest that TnT plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction. Isothermal titration calorimetry revealed that unlike vertebrate TnTs, Pifuc-TnT does not interact with P. fucata tropomyosin-1 nor with tropomyosin-2. These findings in P. fucata imply that Tn functions differently in molluscan muscle than it does in vertebrates.展开更多
Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralizatio...Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.展开更多
In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process, All these activities are very energy...In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process, All these activities are very energy intensive; therefore, investigations of the energy metabolism pathways of different oyster tissues will facilitate understanding of oyster biomineralization physiology. A full-length cDNA encoding the F1- ATPase beta-subunit （the F1-β-subunit, a major calalytic subunit of F-ATPase） from the pearl oyster （Pinctada fucata） was cloned using the homology strategy with a pair of degenerated primers based on the conserved regions of other animals＇ F1-β-subunit genes. Sequencing and structural analyses showed that the obtained sequence shared high identity with other animals＇ F1-β-subunits, and had a unique phosphorylation site of PKC and CK II on the external surface of the putative protein. Results from semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization demonstrated this oyster F1-β-subunit mRNA is abundant in the gill and mantle, and distributed widely in the periostracal groove, the outer folder. and the dorsal region of the mantle and in the gill epithelial cells. These tissues were the main regions that participate in biomineralization processes such as calcium uptake, transport, and matrix secretion. The results indicate that tissues involved in biomineralization have stronger energy metabolic processes and that F1-ATPase might play an important role in oyster biomineralization by providing energy transport.展开更多
Plasma membrane calcium ATPase （PMCA） plays a critical role in transporting Ca^2＋ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2＋ homeostasis and in biominerali...Plasma membrane calcium ATPase （PMCA） plays a critical role in transporting Ca^2＋ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2＋ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further understand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca^2＋ homeostasis and the mechanism of mineralization in pearl oyster.展开更多
基金supported by the Science and Technology Program of Guangdong Province (No.2022A1515010030)the National Natural Science Foundation of China (No.32102817)+3 种基金the Program for Sientific Research Start-up Funds of Guangdong Ocean University (No.060302022304)the Department of Education of Guangdong Province (Nos.2020ZDZX1045 and 2021KCXTD026)the Earmarked Fund for CARS-49the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (No.2023KJ146)。
文摘The yellow-colored line of pearl oyster Pinctada fucata martensii displays a yellow prismatic layer and a white nacreous layer that can be used as an ideal model for research on shell color formation.Micro-Raman spectroscopy and transcriptome analyses were performed to explore the potential molecular mechanism underlying the phenotype differentiation.The micro-Raman spectroscopy results indicate that the prismatic layer exhibits distinct characteristic peaks of carotenoids,while these peaks are not prominent in the nacreous layer.In the transcriptome comparison of the central zone of mantle and mantle edge tissue,which function in nacreous and prismatic layer formation,respectively,935 significantly differentially expressed genes(DEGs)were identified,with 385 genes upregulated and 227 genes downregulated(|log_(2)(Fold change)|>1 and false discovery rate<0.05)in the mantle edge tissue.Among these genes,some were associated with melanoma/melanogenesis,such as tyrosinase,zinc metalloprotease,glutathione S-transferase,and ATP-binding cassette sub-family;some were associated with the carotenoid-related pathway,including scavenger receptors,cytochrome P450 and lipoprotein receptor.Genes associated with porphyrin metabolism,including porphobilinogen deaminase,and copper/zinc superoxide dismutase,and genes associated with shell matrix protein,including amorphous calcium carbonate binding protein,shematrin,PIF,and collagen,also exhibited significantly different expressions.It is speculated that the different colours between prismatic layer and nacreous layer in the yellow-colored line of P.f.martensii might be resulted from melanin,carotenoids and porphyrin metabolism,while genes related to shell structure and biomineralization might also affect coloration.Our results provide new insights to understand the mechanism of shell color formation in mollusca.
基金Supported by the Natural Science Foundation of Guangdong Province,China(No.2019A1515011968)the Key Special Project for Introduced Talents Team of the Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou)(No.GML2019ZD0401)+1 种基金the Earmarked Fund for the Modern Agro-industry Technology Research System(No.CARS-49)the Science and Technology Planning Project of Guangdong Province,China(No.2020B1212060058)。
文摘Inhibitory Smads(I-Smads),which belong to the Smad family and inhibit bone morphogenic protein 2(BMP2)signaling by a variety of mechanisms,can suppress innate immunity responses in vertebrates.However,there are no reports for the role of Smad6 in immunity in mollusks.In this study,we showed that Smad6 of the pearl oyster Pinctada fucata martensii was located in the Smad6 cluster of the phylogenetic tree;mRNA expression of Smad6 and Smad3 was up-regulated after lipopolysaccharide and polyinosinic:polycytidylic challenge;and transcript levels of Smad6 and Smad3 showed opposite patterns during wound healing.Under salinity stress,water inflow and outflow in the gills appear to be regulated by BMP2-Smads signals,and BMP2-Smads signaling may be closely related to the immune response.Our results indicate that Smad6 is involved in immunity,that it plays a positive role in the response to immune challenge and an inhibitory role during wound healing,and that Smad6 and Smad3 may work against each other.
基金supported by the Earmarked Fund for the China Agriculture Research System(No.CARS-49)the Science and Technology Planning Project of Guang-dong Province,China(No.No2020B1212060058).
文摘The pearl oyster Pinctada fucata martensii is an economically valuable shellfish that is cultured for seawater pearl pro-duction,which mainly depends on oyster growth.However,the growth mechanisms of the pearl oyster are still poorly understood.In this study,oysters were grouped with relative growth rate,including fast-growing(FG)group and slow-growing(SG)group.Oxford Nanopore Technologies(ONT)long-read sequencing was applied to investigate the molecular mechanisms involved in the growth of this species.Five alternative splicing(AS)types were analyzed in both FG and SG groups,which include alternative 3’splice site,alternative 5’splice site,exon skipping,intron retention,and mutually exclusive exon.Transcriptome analysis showed that four of five different AS events(excluding mutually exclusive exons)occurred more frequently in FG than in SG oysters,and the five main AS types exhibited different characteristics.The AS events that were detected may be involved in growth,and the difference in ex-pression of AS events between FG and SG oysters may be involved in the mechanism underlying the difference in growth.Fifty dif-ferentially expressed genes(DEGs)were identified between the FG and SG oysters.The results showed that 40 genes were signifi-cantly up-regulated in FG oysters,while 10 genes were significantly down-regulated in SG oyster.Several genes related to nutrient metabolism,shell formation,and immunity were more highly expressed in FG oysters than in SG oysters.In summary,FG oysters exhibited higher metabolic and biomineralization activities and had a more powerful immune system than SG oysters.These results provide insight into the growth of P.f.martensii that can be used to improve breeding programs.
基金The research was financially supported by Science and Technology Program of Guangdong Province(Grant No.2021B0202020003,and 2022A1515010030)National Natural Science Foundation of China(Grant No.32102817)+2 种基金Department of Education of Guangdong Province(Grant No.2019KQNCX043,2020ZDZX1045 and 2021KCXTD026)Special promotion of fishery science and technology of Ocean and Fisheries Bureau of Guangdong Province(Grant No.B201601-Z09)the earmarked fund for CARS-49.
文摘Differentially expressed genes(DEGs)between individuals with high(HC)and low(LC)total carotenoid content(TCC)were sampled from a selected line of Pinctada fucata martensii with black shell in the prismatic layer.The expression levels of candidate genes were verified by qRT-PCR.Targeted resequencing was used to detect SNPs in a candidate gene,PmSR-BI.The association of TCC with SNPs in PmSR-BI was determined.Results showed that a total of 1025 DEGs were identified between HC and LC.The expression levels of the candidate gene PmSR-BI in HC were higher than those in LC.Seven SNPs in the exon and eight SNPs in the 5′regulatory regions of PmSR-BI were found.Association analysis showed that one SNP in the exon and two SNPs in the 5′regulatory regions of PmSR-BI were significantly associated with the TCC(P<0.05).All SNPs of PmSR-BI were divided into four blocks.CC haplotype in Block 1 and AG haplotype in Block 3 were significantly higher than other haplotypes.These results help elucidate the mechanism underlying carotenoid metabolism and develop marker-assisted breeding design in the species.
文摘[目的]开发马氏珠母贝(Pinctada fucata)基因组资源,挖掘功能基因。[方法]以马氏珠母贝血细胞为试材,利用单分子实时技术(single-molecule real time,SMRT)进行全长转录组测序,并对所获得unigenes进行功能注释和基因结构分析。[结果]共获得277064条全长非嵌合序列和82381个基因。经过比对nr、SwissProt、KEGG和KOG数据库进行注释和功能分类后,共得到59621个注释基因。同时,在8493条基因中共发现11219个SSR位点,其中以二核苷酸重复基元类型最高(50.9%)。生物信息学分析鉴定得到20013个lncRNA、2004个转录因子、10522个可变剪切分析位点。[结论]使用SMRT技术能够深入挖掘马氏珠母贝全长转录组数据,为进一步探讨马氏珠母贝功能基因的挖掘、免疫响应及遗传机制的研究提供可靠的基因组资源。
基金Supported by the Earmarked Fund for Modern Agro-industry Technology Research System(No.CARS-49)the Marine Fishery Science and Technology Development Project of Guangdong Province,China(Nos.Z2014012,Z2015014)the Science and Technology Planning Project of Guangdong Province,China(No.2014B030301064)
文摘The mechanisms of sex determination and sex differentiation in the pearl oyster P inctada fucata are currently poorly understood. We therefore investigated the roles of orthologs of the D mrt gene family, key players in male gonad differentiation in mammals, in P. fucata sex diff erentiation and sexual development. Pf-Dmrt4 exhibits features typical of the D mrt family, and displays significant homologies to the DMRT4 cluster. Pf-Dmrt4 mRNA expression in the gonads during a gametogenic cycle, measured by quantitative polymerase chain reaction, was maximal in mature individuals. P f-Dmrt4 expression, demonstrated by in situ hybridization, was localized in the spermatozoa, spermatids, oocytes and vitellogenic oocytes. Knockdown of Pf-Dmrt4 with double-stranded RNA resulted in decreased mRNA expression levels. And Pf-Dmrt4-dsRNA-injected groups showed spawning-stage male gonads, with ruptured follicles and released spermatozoa. Our results enhance the understanding of sex determination and differentiation in P. fucata and suggest that Pf-Dmrt4 could be involved in male gonadal development, and maintenance of male gonadal function.
文摘Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that binds Ca2+ and is a thin filament-associated regulator of vertebrate striated muscle contraction. The function of troponin I (TnI) in vertebrates has been extensively characterized, but its role in molluscan muscles has not yet been elucidated. Our previous work suggested that the troponin C subunit has a role in adductor phasic muscle but not in catch muscle. Here, we investigated the molecular characteristics of TnI from the bivalve Japanese pearl oyster, Pinctada fucata to aid the elucidation of the function of molluscan muscle troponin. We determined the primary structure of the full-length TnI protein from the P. fucata adductor muscle (Pifuc-TnI) and found that it is composed of 286 amino acid residues with a predicted molecular weight of 33,737. Motif structure predictions and multiple sequence alignments revealed that Pifuc-TnI has a 138 residue extension at its N-terminus compared with rabbit TnI. This is analogous to characterized TnIs from other mollusks. However, unlike scallop TnI, Pifuc-TnI is predicted to contain two cAMP-dependent protein kinase phosphorylation sites, at residues 39 - 45 (RRGTEDD) and 145 - 151 (KKKSKRK). Phylogenetic analysis indicated that Pifuc-TnI and molluscan TnIs were grouped into the same clade. Pifuc-TnI gene structure predictions using Splign alignment of our obtained cDNA and genome sequences indicated that Pifuc-TnI consists of fifteen exons, with the start and stop codons located in exon 2 and exon 11, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TnI gene is predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, and is not expressed in the gill, mantle or foot. These findings suggest that TnI, as a component of the troponin complex, plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction.
文摘Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has been obscure in molluscan muscles. Our previous work indicated that the TnC and TnI subunits work in adductor phasic muscle, but not in catch muscle. Here, we have characterized TnT from the Japanese bivalve pearl oyster Pinctada fucata to start to explain the function of Tn in molluscan muscle contraction. We determined the primary structure of the full-length TnT protein from the P. fucata adductor muscle (Pifuc-TnT), and found that it is composed of 316 amino acid residues with a predicted molecular mass of 37.4 kDa. Multiple sequence alignment showed that Pifuc-TnT has an extension of >60 residues at the C-terminus that are not present in vertebrate TnTs, including known TnTs from other mollusks. Pifuc-TnT gene structure predictions using Splign alignment of the cDNA generated in this study and genome sequences indicated that Pifuc-TnT consists of 13 exons. Start and stop codons are located in exons 2 and 12, respectively. Quantitative real-time PCR revealed that the Pifuc-TnT gene was predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, slightly in gill, and not at all in mantle and foot. These findings suggest that TnT plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction. Isothermal titration calorimetry revealed that unlike vertebrate TnTs, Pifuc-TnT does not interact with P. fucata tropomyosin-1 nor with tropomyosin-2. These findings in P. fucata imply that Tn functions differently in molluscan muscle than it does in vertebrates.
基金the National Natural Science Foundation of China (Nos. 30371092, 30221003, and 30530600)
文摘Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.
基金Supported by the National High-Tech Research and Development (863) Program of China (No. 2003AA603430)the National Natural Science Foundation of China (No. 30371092)
文摘In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process, All these activities are very energy intensive; therefore, investigations of the energy metabolism pathways of different oyster tissues will facilitate understanding of oyster biomineralization physiology. A full-length cDNA encoding the F1- ATPase beta-subunit （the F1-β-subunit, a major calalytic subunit of F-ATPase） from the pearl oyster （Pinctada fucata） was cloned using the homology strategy with a pair of degenerated primers based on the conserved regions of other animals＇ F1-β-subunit genes. Sequencing and structural analyses showed that the obtained sequence shared high identity with other animals＇ F1-β-subunits, and had a unique phosphorylation site of PKC and CK II on the external surface of the putative protein. Results from semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization demonstrated this oyster F1-β-subunit mRNA is abundant in the gill and mantle, and distributed widely in the periostracal groove, the outer folder. and the dorsal region of the mantle and in the gill epithelial cells. These tissues were the main regions that participate in biomineralization processes such as calcium uptake, transport, and matrix secretion. The results indicate that tissues involved in biomineralization have stronger energy metabolic processes and that F1-ATPase might play an important role in oyster biomineralization by providing energy transport.
基金the National Natural Science Foundation of China (Nos. 30530600, 30371092, and 30221003)
文摘Plasma membrane calcium ATPase （PMCA） plays a critical role in transporting Ca^2＋ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2＋ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further understand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca^2＋ homeostasis and the mechanism of mineralization in pearl oyster.
