采用双向凝胶电泳技术优化分离蓝斑背肛海兔(Notarcus leachii cirrosusStimpson,NLCS)口腔神经节(Buccal Ganglion,BG)蛋白质组,并获得约300个蛋白质斑点.用组合基质辅助激光解吸电离化飞行时间(MALD I-TOF)质谱技术和胶内酶解技术测...采用双向凝胶电泳技术优化分离蓝斑背肛海兔(Notarcus leachii cirrosusStimpson,NLCS)口腔神经节(Buccal Ganglion,BG)蛋白质组,并获得约300个蛋白质斑点.用组合基质辅助激光解吸电离化飞行时间(MALD I-TOF)质谱技术和胶内酶解技术测定BG蛋白质组中的96个蛋白质斑点的肽指纹(Peptide m ass fin-gerprint,PMF)图谱.经数据库检索与比对后,发现96种蛋白质中仅有4种蛋白质可获得较高的匹配率,它们分别是微管蛋白(Tubu lin)、肌动蛋白(Actin)和两个1,5-二磷酸核酮糖-羧化酶/加氧酶(R ibu lose-1,5-b i-sphosphate carboxylase/oxygenase,R ibu lose),均属于神经细胞骨架蛋白质;同时还发现一种交配信号肽前体(Peptide m ating pheromone precursor).利用LOC trees软件和分类法对56种蛋白质进行亚细胞定位与分类.展开更多
The thermoregulatory behavior of sea hare Aplysia californica was determined in a horizontal thermal gradient;with a preferred temperature (PT) of 18.3°C for the day cycle and 20.8°C for the night cycle. The...The thermoregulatory behavior of sea hare Aplysia californica was determined in a horizontal thermal gradient;with a preferred temperature (PT) of 18.3°C for the day cycle and 20.8°C for the night cycle. The displacement velocity demonstrated an initial rate of 30 cm·hˉ1 and gradually the velocity diminished to 18 cm·hˉ1 with several fluctuations mainly at 02:00 am. Critical Temperature Maxima (CTMax refers to the temperature point where at least 50% of the experimental group have a loss of attachment) was measured at three acclimation temperatures (16°C, 19°C and 22°C). At the lowest acclimation temperature (16°C), 50% of the experimental group had an attachment loss at CTMax 32.7°C, and in a higher acclimation temperature (22°C) CTMax was 36.2°C. The Oxygen Consumption Rate (OCR) was closely correlated to acclimation temperature, and at 16°C and 19°C sea hare had a relatively stable metabolic rate, with OCR increasing to 9 mg O2 hˉ1·kgˉ1 w.w. in a higher acclimation temperature.展开更多
选择DEAE-52纤维素和Sephadex G-150为分离介质,采用柱层析法分离纯化海兔卵多肽酶(En-dopeptidease of Aplysiaegg,AEE)。选用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定AEE纯度和亚基分子量(约39kDa)。基质辅助激光解吸/电离...选择DEAE-52纤维素和Sephadex G-150为分离介质,采用柱层析法分离纯化海兔卵多肽酶(En-dopeptidease of Aplysiaegg,AEE)。选用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定AEE纯度和亚基分子量(约39kDa)。基质辅助激光解吸/电离(MALDI-TOF)质谱技术测定AEE由单类型亚基(M)组成,其m/z比值分别为12738.17、18108.79和38221.42,即分子式为[M3+]、[M2+]和[M+]。其中AEE分子量为38221.42Da(称为AEE39),是一种含金属锌的多肽酶。以胰岛素(INS)为探针,研究AEE39酶切INS能力,其酶切产物m/z比值为1449.51、2085.84、4080.41和4165.42。自行设计INS序列分析软件,鉴定AEE39酶切INS的位点,发现易酶切位点是Leu-X(氨基酸残基),其次是Glu-X,部分Phen-X、Asn-X和Ser-X结构也可以发生酶切。通过INS和海兔吸引素的分子结构比对后,指出海兔卵中的AEE39主要功能之一是负责酶切海兔吸引素,起着海兔之间信息交往、召唤、识别和交配的重要作用。AEE39具有酶切酸性多肽(acidic pep-tide,AP)中Leu-Leu的能力,属于一种广谱性多肽内切酶。展开更多
In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a sys...In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a system for the 5’ and 3’ rapid amplification of cDNA ends, we obtained a 1352 bp chitinase gene (AkChi) from the ovotestis of A. kurodai. AkChi contains a 1263 bp open reading frame that encodes 421 amino acids. The domain structure predicted from the deduced amino acid sequence was an N-terminal signal peptide and a catalytic domain of glycoside hydrolase (GH) family 18 chitinase. A comparative analysis of the deduced amino acid sequences of AkChi with those of the acidic mammalian chitinase of the California sea hare Aplysia californica revealed the highest homology at 83%. The purified chitinase from the ovotestis was digested by trypsin, and 119 residues of digested peptides were consistent with the deduced amino acid sequence of AkChi. We used RT-PCR to evaluate the expression of AkChi in various tissues of A. kurodai, and we observed that AkChi was expressed only in the ovotestis. A phylogenetic tree analysis, performed using the amino acid sequences of AkChi and known GH family 18 chitinases, showed that AkChi was separated from the molluscan chitinases with a chitin binding domain. To our knowledge, this is the first study demonstrating the cDNA cloning of an ovotestis chitinase from a sea hare.展开更多
文摘采用双向凝胶电泳技术优化分离蓝斑背肛海兔(Notarcus leachii cirrosusStimpson,NLCS)口腔神经节(Buccal Ganglion,BG)蛋白质组,并获得约300个蛋白质斑点.用组合基质辅助激光解吸电离化飞行时间(MALD I-TOF)质谱技术和胶内酶解技术测定BG蛋白质组中的96个蛋白质斑点的肽指纹(Peptide m ass fin-gerprint,PMF)图谱.经数据库检索与比对后,发现96种蛋白质中仅有4种蛋白质可获得较高的匹配率,它们分别是微管蛋白(Tubu lin)、肌动蛋白(Actin)和两个1,5-二磷酸核酮糖-羧化酶/加氧酶(R ibu lose-1,5-b i-sphosphate carboxylase/oxygenase,R ibu lose),均属于神经细胞骨架蛋白质;同时还发现一种交配信号肽前体(Peptide m ating pheromone precursor).利用LOC trees软件和分类法对56种蛋白质进行亚细胞定位与分类.
文摘The thermoregulatory behavior of sea hare Aplysia californica was determined in a horizontal thermal gradient;with a preferred temperature (PT) of 18.3°C for the day cycle and 20.8°C for the night cycle. The displacement velocity demonstrated an initial rate of 30 cm·hˉ1 and gradually the velocity diminished to 18 cm·hˉ1 with several fluctuations mainly at 02:00 am. Critical Temperature Maxima (CTMax refers to the temperature point where at least 50% of the experimental group have a loss of attachment) was measured at three acclimation temperatures (16°C, 19°C and 22°C). At the lowest acclimation temperature (16°C), 50% of the experimental group had an attachment loss at CTMax 32.7°C, and in a higher acclimation temperature (22°C) CTMax was 36.2°C. The Oxygen Consumption Rate (OCR) was closely correlated to acclimation temperature, and at 16°C and 19°C sea hare had a relatively stable metabolic rate, with OCR increasing to 9 mg O2 hˉ1·kgˉ1 w.w. in a higher acclimation temperature.
文摘选择DEAE-52纤维素和Sephadex G-150为分离介质,采用柱层析法分离纯化海兔卵多肽酶(En-dopeptidease of Aplysiaegg,AEE)。选用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定AEE纯度和亚基分子量(约39kDa)。基质辅助激光解吸/电离(MALDI-TOF)质谱技术测定AEE由单类型亚基(M)组成,其m/z比值分别为12738.17、18108.79和38221.42,即分子式为[M3+]、[M2+]和[M+]。其中AEE分子量为38221.42Da(称为AEE39),是一种含金属锌的多肽酶。以胰岛素(INS)为探针,研究AEE39酶切INS能力,其酶切产物m/z比值为1449.51、2085.84、4080.41和4165.42。自行设计INS序列分析软件,鉴定AEE39酶切INS的位点,发现易酶切位点是Leu-X(氨基酸残基),其次是Glu-X,部分Phen-X、Asn-X和Ser-X结构也可以发生酶切。通过INS和海兔吸引素的分子结构比对后,指出海兔卵中的AEE39主要功能之一是负责酶切海兔吸引素,起着海兔之间信息交往、召唤、识别和交配的重要作用。AEE39具有酶切酸性多肽(acidic pep-tide,AP)中Leu-Leu的能力,属于一种广谱性多肽内切酶。
文摘In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a system for the 5’ and 3’ rapid amplification of cDNA ends, we obtained a 1352 bp chitinase gene (AkChi) from the ovotestis of A. kurodai. AkChi contains a 1263 bp open reading frame that encodes 421 amino acids. The domain structure predicted from the deduced amino acid sequence was an N-terminal signal peptide and a catalytic domain of glycoside hydrolase (GH) family 18 chitinase. A comparative analysis of the deduced amino acid sequences of AkChi with those of the acidic mammalian chitinase of the California sea hare Aplysia californica revealed the highest homology at 83%. The purified chitinase from the ovotestis was digested by trypsin, and 119 residues of digested peptides were consistent with the deduced amino acid sequence of AkChi. We used RT-PCR to evaluate the expression of AkChi in various tissues of A. kurodai, and we observed that AkChi was expressed only in the ovotestis. A phylogenetic tree analysis, performed using the amino acid sequences of AkChi and known GH family 18 chitinases, showed that AkChi was separated from the molluscan chitinases with a chitin binding domain. To our knowledge, this is the first study demonstrating the cDNA cloning of an ovotestis chitinase from a sea hare.
