铁蛋白在虾夷扇贝新品种“海大金贝”高抗逆性中的作用及机理研究
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摘要
1“海大金贝”和普通虾夷扇贝的蛋白组学初步比较分析
与外界环境、病害生物和敌害生物相比，遗传结构是影响扇贝存活率的最根本的因素。为了保证扇贝养殖业的持续健康发展，需改善和培育高抗逆性的扇贝。近年来，分子标记辅助育种工作已被全面开展，对抗逆性分子标记的筛查也越来越多。本文以富含类胡萝卜素且具高抗逆性的虾夷扇贝新品种“海大金贝”为材料，对其高抗性进行了分子机理的探索。本研究运用蛋白组学的方法对“海大金贝”和普通虾夷扇贝雄性性腺中的差异蛋白进行了分析比较，最终得到7个差异蛋白点，即proteasome subunit alpha type-1(PS)，fructose-bisphosphate aldolase(FBA)，peroxisomal3,2-trans-enoyl-CoA isomerase (PECI)，chaperonin containingTCP1(CCT)，ferritin (FTN)，elongation factor1-beta (EF1B)和β-tubulin (TUBB)。同时，Real-time PCR分析表明其中四个基因（PS, FBA, PECI and CCT）的mRNA表达含量及其蛋白的含量在两种虾夷扇贝中是一致的。分析表明：1）这些差异基因参与了各种各样的生物过程，如脂类和糖类代谢过程、蛋白折叠和降解过程。2）类胡萝卜素累积可能影响了PPARs代谢通路，而使上述基因的表达发生了变化。3）铁蛋白FTN在先天免疫系统中的重要作用，可作为研究“海大金贝”高抗逆性的重要候选基因。本研究为海洋软体动物类胡萝卜素累积的研究开启了崭新的一页并奠定了一定的分子基础。
2虾夷扇贝六种铁蛋白亚基的鉴定及特征分析
作为一种重要的铁离子储存蛋白，铁蛋白在维系胞内铁离子平衡和先天防御过程中起着重要的作用。本研究中，成功克隆了6个虾夷扇贝铁蛋白亚基（PyFer1,PyFer2, PyFer3, PyFer4, PyFer5和PyFer6）的cDNA及DNA序列；其中，PyFer1、PyFer2、PyFer3和PyFer4均由4个外显子和3个内含子组成，PyFer5由7个外显子和6个内含子组成，PyFer6则由5个外显子和4个内含子组成。在PyFer4和PyFer6第二个内含子区域，存在小卫星序列。除了PyFer5和PyFer6外，其余4个虾夷扇贝铁蛋白5’-UTR区域都含有铁反应调控元件IRE。与此同时，在PyFer2、PyFer4和PyFer6的3’-UTR区域分别存在3、17和1个A+U mRNA不稳定原件（TATT或ATTTA）。6种铁蛋白亚基ORF长度分别为522、516、516、519、687和663bp，编码173、171、171、172、228和220个氨基酸。这些铁蛋白均含有典型的铁蛋白结构特征，即4个长α螺旋，1个短α螺旋，1个长环。生物信息学预测结果显示，PyFer1、PyFer2、PyFer3和PyFer4不但含有铁氧化酶作用中心（E Y E E H E Q），还含有铁水合作用位点（D E E），而PyFer5仅含有铁氧化酶作用中心，PyFer6并不含有完整的铁氧化酶作用中心。PyFer1、PyFer2、PyFer3和PyFer4理化性质较为相似，属于胞质型可溶蛋白；而PyFer5和PyFer6铁蛋白N端含有信号肽序列，属于分泌型可溶蛋白。定量PCR分析显示，6种铁蛋白亚基在虾夷扇贝D型幼虫期表达量均有显著的提升，在成体虾夷扇贝外套膜和肝胰腺中的表达量较高。虾夷扇贝受铁或者鳗弧菌胁迫后，6种铁蛋白的表达量均有了显著的变化。重组蛋白（rPyFer1、rPyFer2、rPyFer3和rPyFer4）展示了明显的铁氧化酶和抑菌活性。细胞定位显示，铁蛋白广泛分布在虾夷扇贝不同细胞类型中。以上结果表明这6种铁蛋白可能参与了虾夷扇贝多个基础生理活动，如免疫应答、铁离子平衡和贝壳形成过程。
3铁蛋白在“海大金贝”高抗逆性中的作用
作为一种重要的铁离子储存蛋白，铁蛋白参与了虾夷扇贝的免疫应答、铁离子平衡和贝壳形成等多个基础生理活动过程。本实验以普通虾夷扇贝和“海大金贝”为材料，对两者经过铁或鳗弧菌胁迫后6种铁蛋白的反应灵敏度进行了比较，发现与普通虾夷扇贝相比，“海大金贝”中的6种铁蛋白均表现出更敏感快捷的反应，表明铁蛋白可能在“海大金贝”的高抗逆性中起着重要的作用。
1Proteomic analysis identifies proteins related to stress resistance in Yessoscallop (Patinopecten yessoensis)
Compared with outside environment, pathogens, protozoan, and predators, geneticstructure is a most fundamental factor to influence the survival rate of Yesso scallop.In order to keep sustainable development of breeding, it is urgent to improve andbreed a new variety with higher resistance. Most recently, based on molecular markers,like resistant genes, breeding has been promoted and got better results. A new variety,“Haida golden scallop”, with higher content of carotenoids and higher resistancecompared to the common Yesso scallop, has been developed successfully by ourgroup. To unravel the molecular mechanism of high stress resistance in “Haida goldenscallop”, a proteomic approach was applied in this study to explore the differencesbetween the new variety and common individuals. It resulted in7differentiallyexpressed proteins. Real-time PCR showed that four of the corresponding genes werealso significantly up-regulated at the mRNA level in the new variety. These resultsindicate that:1) Genes involved in various biological processes, such as lipid andglucose metabolism, protein-folding and degradation, were altered.2) Carotenoidsaccumulation may influence peroxisome proliferator-activated receptor (PPAR)signaling pathway, and PPARs may play a vital role in these above changes.3)Ferritin, as an important factor in innate immune is a good candidate for unravelingthe mechanism of high stress resistance in “Haida golden scallop”. This studyrepresents a starting point for future work concerning the genetic basis of carotenoid accumulation across all marine mollusks.
2Identification and characterization of six ferritin subunits involved in immunedefense of the Yesso scallop (Patinopecten yessoensis)
As a primary iron storage protein, ferritin plays a vital role in iron homeostasis andinnate immunity. In this study, the DNA and full-length cDNA sequences of sixferritin subunits (PyFer1, PyFer2, PyFer3, PyFer4, PyFer5, and PyFer6) were clonedfrom the Yesso scallop, Patinopecten yessoensis. PyFer1, PyFer2, PyFer3, andPyFer4consist of four exons and three introns；PyFer5is composed of seven exonsand six introns, and PyFer6has a five-exon/four-intron structure. Minisatellite DNAsare found in intron2of PyFer4and PyFer6. Except for PyFer5and PyFer6, the restfour ferritins (PyFer1, PyFer2, PyFer3, and PyFer4) contain putative iron-responseelements (IREs) in their5’ UTRs. Meanwhile, multiple A+U-destabilizing elements(TATT or ATTTA) are present in the3’ UTRs of PyFer2, PyFer4and PyFer6. Theopen reading frames (ORFs) of the six ferritins are522,516,516,687, and663bp inlength, encoding173,171,171,172,228, and220amino acids, respectively. Theseproteins have typical ferritin structures, with four long α-helices, one short α-helix andan L-loop. Four of the predicted proteins (PyFer1, PyFer2, PyFer3, and PyFer4)possess both the ferroxidase center of mammalian H ferritins (E Y E E H E Q) and theiron nucleation site of mammalian L ferritins (D E E), and the four recombinantproteins (rPyFer1, rPyFer2, rPyFer3, and rPyFer4) possess apparent ferroxidaseactivity. Moreover, PyFer5only contains the ferroxidase center, and PyFer6has anincomplete ferroxidase center. PyFer1, PyFer2, PyFer3, and PyFer4are likely tobelong to cytoplasmic ferritins, while both PyFer5and PyFer6possess signal peptides,suggesting that they are secreted ferritins, which is in accordance with the result ofphylogenetic analysis. Quantitative real-time PCR analysis revealed that theexpression of the six PyFers was significantly elevated at the D-shaped stage and wasrelatively high in the adult mantle and hepatopancreas. Furthermore, the six PyFerswere significantly up-regulated by iron or bacterial challenge, and all four purifiedrecombinant PyFers (rPyFer1, rPyFer2, rPyFer3, and rPyFer4) were able to inhibit the growth of the scallop pathogen Vibrio anguillarum. Immunohistochemical analysisshowed that ferritins were widely distributed in various cell types of Yesso scallop.These results suggest that these PyFers are likely to play important roles in manyfundamental biological processes in P. yessoensis, including immune defense, ironhomeostasis, and shell development.
3Role of ferritin in the high sresistance of “Haida golden scallop”
As primary iron storage proteins, the six ferritins have been demonstrated to playvital roles in the immune defense, iron homeostasis, and shell development of P.yessoensis. In this study, relative mRNA expressions of the six PyFers were evaluatedby iron or bacterial challenge between “Haida golden scallop” and the commonindividuals. Results showed that all six ferritin subunits from “Haida golden scallop”were more sensitive to the ferrous iron or V. anguillarum than those from the commonindividuals, indicating that ferritins might participate in the high resistance of “Haidagolden scallop”.
与外界环境、病害生物和敌害生物相比，遗传结构是影响扇贝存活率的最根本的因素。为了保证扇贝养殖业的持续健康发展，需改善和培育高抗逆性的扇贝。近年来，分子标记辅助育种工作已被全面开展，对抗逆性分子标记的筛查也越来越多。本文以富含类胡萝卜素且具高抗逆性的虾夷扇贝新品种“海大金贝”为材料，对其高抗性进行了分子机理的探索。本研究运用蛋白组学的方法对“海大金贝”和普通虾夷扇贝雄性性腺中的差异蛋白进行了分析比较，最终得到7个差异蛋白点，即proteasome subunit alpha type-1(PS)，fructose-bisphosphate aldolase(FBA)，peroxisomal3,2-trans-enoyl-CoA isomerase (PECI)，chaperonin containingTCP1(CCT)，ferritin (FTN)，elongation factor1-beta (EF1B)和β-tubulin (TUBB)。同时，Real-time PCR分析表明其中四个基因（PS, FBA, PECI and CCT）的mRNA表达含量及其蛋白的含量在两种虾夷扇贝中是一致的。分析表明：1）这些差异基因参与了各种各样的生物过程，如脂类和糖类代谢过程、蛋白折叠和降解过程。2）类胡萝卜素累积可能影响了PPARs代谢通路，而使上述基因的表达发生了变化。3）铁蛋白FTN在先天免疫系统中的重要作用，可作为研究“海大金贝”高抗逆性的重要候选基因。本研究为海洋软体动物类胡萝卜素累积的研究开启了崭新的一页并奠定了一定的分子基础。
2虾夷扇贝六种铁蛋白亚基的鉴定及特征分析
作为一种重要的铁离子储存蛋白，铁蛋白在维系胞内铁离子平衡和先天防御过程中起着重要的作用。本研究中，成功克隆了6个虾夷扇贝铁蛋白亚基（PyFer1,PyFer2, PyFer3, PyFer4, PyFer5和PyFer6）的cDNA及DNA序列；其中，PyFer1、PyFer2、PyFer3和PyFer4均由4个外显子和3个内含子组成，PyFer5由7个外显子和6个内含子组成，PyFer6则由5个外显子和4个内含子组成。在PyFer4和PyFer6第二个内含子区域，存在小卫星序列。除了PyFer5和PyFer6外，其余4个虾夷扇贝铁蛋白5’-UTR区域都含有铁反应调控元件IRE。与此同时，在PyFer2、PyFer4和PyFer6的3’-UTR区域分别存在3、17和1个A+U mRNA不稳定原件（TATT或ATTTA）。6种铁蛋白亚基ORF长度分别为522、516、516、519、687和663bp，编码173、171、171、172、228和220个氨基酸。这些铁蛋白均含有典型的铁蛋白结构特征，即4个长α螺旋，1个短α螺旋，1个长环。生物信息学预测结果显示，PyFer1、PyFer2、PyFer3和PyFer4不但含有铁氧化酶作用中心（E Y E E H E Q），还含有铁水合作用位点（D E E），而PyFer5仅含有铁氧化酶作用中心，PyFer6并不含有完整的铁氧化酶作用中心。PyFer1、PyFer2、PyFer3和PyFer4理化性质较为相似，属于胞质型可溶蛋白；而PyFer5和PyFer6铁蛋白N端含有信号肽序列，属于分泌型可溶蛋白。定量PCR分析显示，6种铁蛋白亚基在虾夷扇贝D型幼虫期表达量均有显著的提升，在成体虾夷扇贝外套膜和肝胰腺中的表达量较高。虾夷扇贝受铁或者鳗弧菌胁迫后，6种铁蛋白的表达量均有了显著的变化。重组蛋白（rPyFer1、rPyFer2、rPyFer3和rPyFer4）展示了明显的铁氧化酶和抑菌活性。细胞定位显示，铁蛋白广泛分布在虾夷扇贝不同细胞类型中。以上结果表明这6种铁蛋白可能参与了虾夷扇贝多个基础生理活动，如免疫应答、铁离子平衡和贝壳形成过程。
3铁蛋白在“海大金贝”高抗逆性中的作用
作为一种重要的铁离子储存蛋白，铁蛋白参与了虾夷扇贝的免疫应答、铁离子平衡和贝壳形成等多个基础生理活动过程。本实验以普通虾夷扇贝和“海大金贝”为材料，对两者经过铁或鳗弧菌胁迫后6种铁蛋白的反应灵敏度进行了比较，发现与普通虾夷扇贝相比，“海大金贝”中的6种铁蛋白均表现出更敏感快捷的反应，表明铁蛋白可能在“海大金贝”的高抗逆性中起着重要的作用。
1Proteomic analysis identifies proteins related to stress resistance in Yessoscallop (Patinopecten yessoensis)
Compared with outside environment, pathogens, protozoan, and predators, geneticstructure is a most fundamental factor to influence the survival rate of Yesso scallop.In order to keep sustainable development of breeding, it is urgent to improve andbreed a new variety with higher resistance. Most recently, based on molecular markers,like resistant genes, breeding has been promoted and got better results. A new variety,“Haida golden scallop”, with higher content of carotenoids and higher resistancecompared to the common Yesso scallop, has been developed successfully by ourgroup. To unravel the molecular mechanism of high stress resistance in “Haida goldenscallop”, a proteomic approach was applied in this study to explore the differencesbetween the new variety and common individuals. It resulted in7differentiallyexpressed proteins. Real-time PCR showed that four of the corresponding genes werealso significantly up-regulated at the mRNA level in the new variety. These resultsindicate that:1) Genes involved in various biological processes, such as lipid andglucose metabolism, protein-folding and degradation, were altered.2) Carotenoidsaccumulation may influence peroxisome proliferator-activated receptor (PPAR)signaling pathway, and PPARs may play a vital role in these above changes.3)Ferritin, as an important factor in innate immune is a good candidate for unravelingthe mechanism of high stress resistance in “Haida golden scallop”. This studyrepresents a starting point for future work concerning the genetic basis of carotenoid accumulation across all marine mollusks.
2Identification and characterization of six ferritin subunits involved in immunedefense of the Yesso scallop (Patinopecten yessoensis)
As a primary iron storage protein, ferritin plays a vital role in iron homeostasis andinnate immunity. In this study, the DNA and full-length cDNA sequences of sixferritin subunits (PyFer1, PyFer2, PyFer3, PyFer4, PyFer5, and PyFer6) were clonedfrom the Yesso scallop, Patinopecten yessoensis. PyFer1, PyFer2, PyFer3, andPyFer4consist of four exons and three introns；PyFer5is composed of seven exonsand six introns, and PyFer6has a five-exon/four-intron structure. Minisatellite DNAsare found in intron2of PyFer4and PyFer6. Except for PyFer5and PyFer6, the restfour ferritins (PyFer1, PyFer2, PyFer3, and PyFer4) contain putative iron-responseelements (IREs) in their5’ UTRs. Meanwhile, multiple A+U-destabilizing elements(TATT or ATTTA) are present in the3’ UTRs of PyFer2, PyFer4and PyFer6. Theopen reading frames (ORFs) of the six ferritins are522,516,516,687, and663bp inlength, encoding173,171,171,172,228, and220amino acids, respectively. Theseproteins have typical ferritin structures, with four long α-helices, one short α-helix andan L-loop. Four of the predicted proteins (PyFer1, PyFer2, PyFer3, and PyFer4)possess both the ferroxidase center of mammalian H ferritins (E Y E E H E Q) and theiron nucleation site of mammalian L ferritins (D E E), and the four recombinantproteins (rPyFer1, rPyFer2, rPyFer3, and rPyFer4) possess apparent ferroxidaseactivity. Moreover, PyFer5only contains the ferroxidase center, and PyFer6has anincomplete ferroxidase center. PyFer1, PyFer2, PyFer3, and PyFer4are likely tobelong to cytoplasmic ferritins, while both PyFer5and PyFer6possess signal peptides,suggesting that they are secreted ferritins, which is in accordance with the result ofphylogenetic analysis. Quantitative real-time PCR analysis revealed that theexpression of the six PyFers was significantly elevated at the D-shaped stage and wasrelatively high in the adult mantle and hepatopancreas. Furthermore, the six PyFerswere significantly up-regulated by iron or bacterial challenge, and all four purifiedrecombinant PyFers (rPyFer1, rPyFer2, rPyFer3, and rPyFer4) were able to inhibit the growth of the scallop pathogen Vibrio anguillarum. Immunohistochemical analysisshowed that ferritins were widely distributed in various cell types of Yesso scallop.These results suggest that these PyFers are likely to play important roles in manyfundamental biological processes in P. yessoensis, including immune defense, ironhomeostasis, and shell development.
3Role of ferritin in the high sresistance of “Haida golden scallop”
As primary iron storage proteins, the six ferritins have been demonstrated to playvital roles in the immune defense, iron homeostasis, and shell development of P.yessoensis. In this study, relative mRNA expressions of the six PyFers were evaluatedby iron or bacterial challenge between “Haida golden scallop” and the commonindividuals. Results showed that all six ferritin subunits from “Haida golden scallop”were more sensitive to the ferrous iron or V. anguillarum than those from the commonindividuals, indicating that ferritins might participate in the high resistance of “Haidagolden scallop”.
引文
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