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Identification and characterization of the calcineurin B in Fenneropenaeus chinensis: its implications on competitive ability

  • Corresponding author: Sheng LUAN, luangsheng@ysfri.ac.cn
  • Received Date: 2019-01-11
    Accepted Date: 2019-04-10
  • Competitive behavior exists widely in animal and plant groups. When groups or individuals have the same needs for the limited resources, competition will occur in intra-species or inter-species. The existence of competitive behavior can significantly affect the growth performance of individuals. There is strong competitive behavior in the breeding of Fenneropenaeus chinensis, and we has proved that the competitive behavior among individuals of F. chinensis has a significant effect on individual growth traits, and this effect can be inherited. However, little information was available for the molecular mechanism of competition behavior in shrimp. Candidate genes involved in competitive behavior of F. chinensis have been identified by comparative transcriptome analysis in our previous study, among which there is a calcineurin (CN) B gene. CN is a highly conserved Ca2+/calmodulin (CaM)-dependent serine/threonine phosphatase, which composes of a catalytic subunit (CNA) and a regulatory subunit (CNB). Previous studies reported that CN-B played an important role in the central nervous system mediated by Ca2+/CaM. In order to further prove the role of CN-B gene in the competitive behavior, in the present study, the full-length cDNA of CN-B of F. chinensis (FcCN-B) was cloned by RACE technology, and its expression in different tissues (nervous, heart, stomach, hepatopancreas, and intestine) between high competitive group (HCG) and low competitive group (LCG) was analyzed by Real-time PCR. The results show that the full-length cDNA of FcCN-B is 2867 bp, containing 95 bp of 5′ untranslated regions (UTR), 540 bp of open reading frame (ORF), and 2232 bp of 3′ UTR. There are four conserved EF-hand Ca2+ binding domains in the ORF of FcCN-B. Protein homology analysis indicated that the amino acid sequence of FcCN-B has high homology with other species (78.8%-93.8%), among which FcCN-B has the highest homology with Eriocheir sinensis (93.8%) and then with Drosophila. melanogaster (90.5%). The phylogenetic relationship analysis showed that vertebrates and invertebrates were independently clustered into two branches. Furthermore, F. chinensis was clustered with E. sinensis into an independent branches, and then it was clustered to D. melanogaster, which suggesting that FcCN-B might have similar functions as it did in D. melanogaster. The results of Real-time PCR showed that the expression of FcCN-B in the nerve ganglion was significantly higher in HCG than in LCG, but its expression in heat was significantly lower in HCG than in LCG. This study preliminarily proved that the calcineurin B may play a certain role in the competition behavior of F. chinensis, and will lay an important foundation for investigating the molecular mechanism of the competition behavior of F. chinensis.
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  • [1] 房继明. 动物行为学知识简介(三): —动物的竞争行为[J]. 生物学通报, 1992(3): 28-30.Fang J M. Introduction of animal behavior knowledge (3) - Competitive behavior of animals[J]. Bulletin of Biology, 1992(3): 28-30(in Chinese).
    [2] 仲伟鹏, 罗坤, 孟宪红, 等. 限制投喂环境下中国对虾体重的间接遗传效应分析[J]. 中国水产科学, 2018, 25(6): 1245-1251.Zhong W P, Luo K, Meng X H, et al. An analysis of the indirect genetic effect on the body weight of Fenneropenaeus chinensis under restrictive feeding conditions[J]. Journal of Fishery Sciences of China, 2018, 25(6): 1245-1251(in Chinese).
    [3] 张沛东, 张秀梅, 李健, 等. 中国明对虾、凡纳滨对虾仔虾的行为观察[J]. 水产学报, 2008, 32(2): 223-228.Zhang P D, Zhang X M, Li J, et al. Observation of behavior in Fenneropenaeus chinensis and Litopenaeus vannamei postlarvae[J]. Journal of Fisheries of China, 2008, 32(2): 223-228(in Chinese).
    [4] 谢保胜, 徐宁迎. 应用动物模型REML法估计金华猪繁殖性状遗传参数[J]. 畜牧与兽医, 2003, 35(2): 6-9. doi: 10.3969/j.issn.0529-5130.2003.02.003Xie B S, Xu N Y. Estimation on genetic parameter of reproductive traits of Jinhua sows by animal mode procedures adopting REML Method[J]. Animal Husbandry & Veterinary Medicine, 2003, 35(2): 6-9(in Chinese). doi: 10.3969/j.issn.0529-5130.2003.02.003
    [5] 夏宣炎, 熊远著. 应用动物模型BLUP方法估计猪个体育种值研究[J]. 华中农业大学学报, 2000(19): 142-146.Xia X Y, Xiong Y Z. Studies on Predicting Breeding Value of Swine Applying BLUP-animal Model[J]. Journal of Huazhong Agricultural University, 2000(19): 142-146(in Chinese).
    [6] 王勇强, 张沅, 张勤, 等. ML法和REML法方差组分估计效率的Monte Carlo模拟研究[J]. 黄牛杂志, 1998, 24(3): 9-12.Wang Y Q, Zhang R, Zhang Q, et al. The simulation study on the effect of ML and REML methods used for variance components estimation[J]. Journal of Yellow Cattle Science, 1998, 24(3): 9-12(in Chinese).
    [7] 李玉华. 应用动物模型BLUP和REML对大白猪遗传评定和遗传参数估计[D]. 泰安: 山东农业大学, 2003.Li Y H. Genetic evaluation and genetic parameter estimation in largewhite pigs using Animal Model BLUP and REML[D]. Taian: Shandong Agricultural University, 2003(in Chinese).
    [8] 刘泽辉. 天府肉羊部分生产性状的遗传参数估计及生长规律研究[D]. 雅安: 四川农业大学, 2006.Liu Z H. Estimation of genetic parameters of production traits and growth rules of Tianfu goat[D]. Ya’an: Sichuan Agricultural University, 2006(in Chinese).
    [9] Luan S, Luo K, Chai Z, et al. An analysis of indirect genetic effects on adult body weight of the Pacific white shrimp Litopenaeus vannamei at low rearing density[J]. Genetics Selection Evolution, 2015, 47: 95. doi: 10.1186/s12711-015-0164-y
    [10] Giri P R, Marietta C A, Higuchi S, et al. Molecular and phylogenetic analysis of calmodulin-dependent protein phosphatase (calcineurin) catalytic subunit genes[J]. DNA and Cell Biology, 1992, 11(5): 415-24. doi: 10.1089/dna.1992.11.415
    [11] Shi X Y, Sun Y, Wang P, et al. The interaction between calcineurin and α-synuclein is regulated by calcium and calmodulin[J]. Biochemical and Biophysical Research Communications, 2018, 496(4): 1109-1114. doi: 10.1016/j.bbrc.2018.01.148
    [12] Rao A. Signaling to gene expression: Calcium, calcineurin and NFAT[J]. Nature Immunology, 2009, 10(1): 3-5. doi: 10.1038/ni0109-3
    [13] Schulz R A, Yutzey K E. Calcineurin signaling and NFAT activation in cardiovascular and skeletal muscle development[J]. Developmental Biology, 2004, 266(1): 1-16.
    [14] Schiaffino S, Serrano A. Calcineurin signaling and neural control of skeletal muscle fiber type and size[J]. Trends in Pharmacological Sciences, 2002, 23(12): 569-575. doi: 10.1016/S0165-6147(02)02111-9
    [15] Olson E N, Williams R S. Calcineurin signaling and muscle remodeling[J]. Cell, 2000, 101(7): 689-692. doi: 10.1016/S0092-8674(00)80880-6
    [16] Wang H G, Pathan N, Ethell I M, et al. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD[J]. Science, 1999, 284(5412): 339-343. doi: 10.1126/science.284.5412.339
    [17] Shenkman B S, Nemirovskaya T L. Calcium-dependent signaling mechanisms and soleus fiber remodeling under gravitational unloading[J]. Journal of Muscle Research and Cell Motility, 2008, 29(6-8): 221-230. doi: 10.1007/s10974-008-9164-7
    [18] Rusnak F, Mertz P. Calcineurin: Form and function[J]. Physiological Reviews, 2000, 80(4): 1483-1521. doi: 10.1152/physrev.2000.80.4.1483
    [19] Kincaid R. Calmodulin-dependent protein phosphatases from microorganisms to man. A study in structural conservatism and biological diversity[J]. Advances in Second Messenger and Phosphoprotein Research, 1993, 27: 1-23.
    [20] Li S, Jia Z R, Li X J, et al. Calmodulin is a stress and immune response gene in Chinese mitten crab Eriocheir sinensis[J]. Fish & Shellfish Immunology, 2014, 40(1): 120-128.
    [21] Li S, Jia Z R, Chen X L, et al. Identification and characterization of the cDNAs encoding the two subunits of Chinese mitten crab (Eriocheir sinensis) calcineurin: Their implications in stress and immune response[J]. Fish & Shellfish Immunology, 2015, 43(1): 91-102.
    [22] Zhang Q L, Li F H, Zhang X J, et al. cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis[J]. Fish & Shellfish Immunology, 2008, 24(5): 584-591.
    [23] Bai H, Du J F, Hu M, et al. Analysis of mechanisms of resistance and tolerance of Escherichia coli to enrofloxacin[J]. Annals of Microbiology, 2012, 62(1): 293-298. doi: 10.1007/s13213-011-0260-3
    [24] Schuh K, Uldrijan S, Gambaryan S, et al. Interaction of the plasma membrane Ca2+ pump 4b/CI with the Ca2+/calmodulin-dependent membrane-associated kinase CASK[J]. Journal of Biological Chemistry, 2003, 278(11): 9778-9783. doi: 10.1074/jbc.M212507200
    [25] Rougier J S, Abriel H. 0151: Role a calcium/calmodulin-dependent serine protein kinase on Cav1.2 channel regulation[J]. Archives of Cardiovascular Diseases Supplements, 2015, 7(2): 167-168.
    [26] Warren W D, Phillips A M, Howells A J. Drosophila melanogaster contains both X-linked and autosomal homologues of the gene encoding calcineurin B[J]. Gene, 1996, 177(1-2): 149-153. doi: 10.1016/0378-1119(96)00291-0
    [27] Batters C, Veigel C. Mechanics and activation of unconventional myosins[J]. Traffic, 2016, 17(8): 860-871. doi: 10.1111/tra.12400
    [28] Polovkova O G, Makeeva O A, Lezhnev A A, et al. Expression profiles of calcineurin pathway genes in myocardium in relation to ischemic heart remodeling in humans[J]. Molecular Biology, 2013, 47(3): 382-388. doi: 10.1134/S0026893313030102
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Identification and characterization of the calcineurin B in Fenneropenaeus chinensis: its implications on competitive ability

    Corresponding author: Sheng LUAN, luangsheng@ysfri.ac.cn
  • 1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
  • 2. Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture and Rural Areas, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
  • 3. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China

Abstract: Competitive behavior exists widely in animal and plant groups. When groups or individuals have the same needs for the limited resources, competition will occur in intra-species or inter-species. The existence of competitive behavior can significantly affect the growth performance of individuals. There is strong competitive behavior in the breeding of Fenneropenaeus chinensis, and we has proved that the competitive behavior among individuals of F. chinensis has a significant effect on individual growth traits, and this effect can be inherited. However, little information was available for the molecular mechanism of competition behavior in shrimp. Candidate genes involved in competitive behavior of F. chinensis have been identified by comparative transcriptome analysis in our previous study, among which there is a calcineurin (CN) B gene. CN is a highly conserved Ca2+/calmodulin (CaM)-dependent serine/threonine phosphatase, which composes of a catalytic subunit (CNA) and a regulatory subunit (CNB). Previous studies reported that CN-B played an important role in the central nervous system mediated by Ca2+/CaM. In order to further prove the role of CN-B gene in the competitive behavior, in the present study, the full-length cDNA of CN-B of F. chinensis (FcCN-B) was cloned by RACE technology, and its expression in different tissues (nervous, heart, stomach, hepatopancreas, and intestine) between high competitive group (HCG) and low competitive group (LCG) was analyzed by Real-time PCR. The results show that the full-length cDNA of FcCN-B is 2867 bp, containing 95 bp of 5′ untranslated regions (UTR), 540 bp of open reading frame (ORF), and 2232 bp of 3′ UTR. There are four conserved EF-hand Ca2+ binding domains in the ORF of FcCN-B. Protein homology analysis indicated that the amino acid sequence of FcCN-B has high homology with other species (78.8%-93.8%), among which FcCN-B has the highest homology with Eriocheir sinensis (93.8%) and then with Drosophila. melanogaster (90.5%). The phylogenetic relationship analysis showed that vertebrates and invertebrates were independently clustered into two branches. Furthermore, F. chinensis was clustered with E. sinensis into an independent branches, and then it was clustered to D. melanogaster, which suggesting that FcCN-B might have similar functions as it did in D. melanogaster. The results of Real-time PCR showed that the expression of FcCN-B in the nerve ganglion was significantly higher in HCG than in LCG, but its expression in heat was significantly lower in HCG than in LCG. This study preliminarily proved that the calcineurin B may play a certain role in the competition behavior of F. chinensis, and will lay an important foundation for investigating the molecular mechanism of the competition behavior of F. chinensis.

  • 动植物群体中广泛存在竞争行为,当群体或个体之间对有限的共同资源有相同需要时,就会发生种内或种间竞争[1]。竞争行为的存在,可显著地影响个体的生长性能[2]。植物在生长过程中,需要争取更有利的光照、水分、养分等,在同一环境下,株形紧凑和根系密度大的植株,能够吸收更多的光照、水分和养分,产量更高;动物在生长和繁衍的过程中,均需要保护好食物、生活空间、巢穴及配偶,攻击性强的动物个体,能摄食更多的饲料、侵占更多的空间和其他资源,生长繁殖和存活性能强[2]

    中国明对虾(Fenneropenaeus chinensis)肉质鲜美、个体大,具有较高的经济价值,是我国重要的养殖虾类。然而中国明对虾表现为运动活跃、攻击频率高、攻击力强、防御反应强烈、容易残食的现象,不适合高密度集约化养殖,严重影响了其产业的发展[3]。通过最大似然法(restricted maximum likelihood method,REML)[4-6]和最佳线性无偏预测(best linear unbiased prediction,BLUP)法[7-9]的遗传评估,已明确凡纳滨对虾个体间的竞争行为对个体生长性状有较显著的影响,且此影响可遗传[9]。因此,获得与竞争行为相关的基因,为通过改良中国明对虾的竞争能力而提高其生长性能提供理论依据和分子基础,对解析竞争行为的发生机理具有重要的科学意义。

    然而,目前尚不清楚中国明对虾竞争行为产生的分子机理,在竞争过程中发挥作用的基因有哪些。为了鉴定出参与中国明对虾竞争过程中的关键基因,前期设置不同的竞争环境刺激的处理组开展了比较转录组分析,筛选获得了批量候选基因,其中包括钙调神经磷酸酶(calcineurin, CN)B基因。CN是一种高度保守的Ca2+/钙调蛋白依赖性磷酸酶,是由催化亚基(CN-A)和调控亚基(CN-B)按1∶1比例组成的异二聚体蛋白,CN-B和CaM都是EF-hand Ca2+结合蛋白质[10]。研究表明,CN-B在神经退行性疾病、细胞骨架稳定、长期记忆、神经突延长等一系列细胞过程中通过Ca2+依赖性信号转导通路发挥重要作用[11-13]。除了在神经系统发挥作用外,CN-B也在调节心脏发育中发挥重要的作用[13],包括肌纤维表型转化、结构重塑和细胞凋亡[14-17]

    CN基因广泛分布于哺乳动物中,在每个组织中都有表达[10],Li shuo等[18] 发现CN尤其在神经细胞和T淋巴细胞中含量丰富,并且在系统发育上高度保守[10, 19]。在水产动物中,目前已有报道显示CN存在于鲎(Limulidae)和中华绒螯蟹(Eriocheir sinensis)的神经组织中[20],而且在中华绒螯蟹中,CN-B在神经节中的表达量高于其他组织[21]。然而,还缺乏CN在中国明对虾中的相关报道。本实验基于转录组测序获得的部分CN-B参考序列,通过RACE技术克隆中国明对虾CN-B基因的cDNA全长序列,并利用real-time PCR(RT-PCR)技术分析其在竞争能力差异显著的个体间的不同组织中的表达情况,为探究该基因在中国明对虾竞争过程中的作用奠定一定的基础。

    • 实验在农业农村部海水养殖遗传育种中心及黄海水产研究所进行。CN基因克隆的实验材料取自健康的中国明对虾G13代核心育种群体中5个个体的肝胰腺和肌肉组织。定量分析的实验群体为中国明对虾G13代核心育种群体,对来自核心群体的100个家系进行了为期4个月的竞争行为能力测试,测试结束后利用REML和BLUP法对该性状进行了遗传评估。通过遗传评估获得可反映每个家系竞争能力强弱的间接遗传效应(indirect genetic effect, IGE)值,IGE值越高表明该家系竞争能力则越弱,反之IGE值越低表明该家系竞争能力则越强[9]。根据参与测试的每个家系的IGE值从高到低的排序,选取3个IGE值最高的家系作为竞争能力弱组(low competitive group, LCG)和3个IGE值最低家系作为竞争能力强组(high competitive group, HCG)用于后续的CN-B基因表达分析,详细信息见表1

      家系类型 fmily type家系名称 family name家系IGE值 family IGE Value平均体重/g average weight
      竞争强家系 high competitive group (HCG) HCG-1 −0.047 9.400
      HCG-2 −0.042 10.466
      HCG-3 −0.040 11.202
      竞争弱家系 low competitive group (LCG) LCG-1 0.031 8.331
      LCG-2 0.033 8.082
      LCG-3 0.051 7.486

      Table 1.  The information for the experimental families of F. chinensis

    • 取样前6个家系分别单独饲养并饥饿2 d。取样分为3组,分别为HCG-1 & LCG-1,HCG-2 & LCG-2和HCG-3 & LCG-3,3个组独立取样,取样时同一组内每个家系选取30尾中国明对虾混合,并投放少量的饲料以此制造较大程度的抢食环境。每组开始抢食后(投饵后10 min),观察肠道饱食程度,抢食能力强的中国明对虾肠道一般比较饱满,抢食能力弱的中国明对虾其肠道相对较空。从HCG中选取抢食能力突出并且肠道饱食的竞争力强的5尾中国明对虾,LCG中选取抢食落后肠道内无饲料或少量饲料的竞争力弱的5尾中国明对虾,分别按照1尾强1尾弱的顺序迅速地取每尾中国明对虾的神经节、心脏、胃、肝胰腺和肠等5种组织速冻于液氮保存,用于后续的实时荧光定量表达分析。

    • 通过TRizol法提取来自中国明对虾5种组织的总RNA ,并通过琼脂糖凝胶电泳检测RNA的完整性和质量。使用NanoDrop 2000、UV/Vis分光光度计(Thermo Fisher Scientific)在260和280 nm的波长下通过分光光度法定量总RNA。根据说明书,使用试剂盒HiScript II Q R T SuperMix for qPCR逆转录酶(Vazyme)和衔接子寡聚(dT)引物,从每个个体5种组织中提取的总RNA(100 μg/μL)分别用于合成相应的cDNA。

    • 基于转录组测序获得的CN-B的核心参考序列,利用Primer Premier 5.0软件设计上下游引物C-F和C-R(表2),验证该序列的准确性。PCR反应程序:98 °C预变性3 min;98 °C变性10 s,57 °C退火30 s,72 °C延伸40 s,35个循环;72 °C最终延伸10 min。反应结束后进行琼脂糖凝胶电泳检测,确定片段大小无误后将目的片段按照胶回收试剂盒说明书进行回收纯化,纯化后的PCR产物连接到pMD18T上,转化后对阳性克隆送交青岛派森诺基因生物技术有限公司测序。

      克隆片段 cloning fragment引物名称 primer name引物序列 primer sequence
      3′RACE C287-2 AATGGAAAATGTAGGTTATTGGGCTC
      C287-2.5 GATCTTAGAGTCTCTTGGCTTTCCCTC
      UMP 5′TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT -3′
      5′RACE B386-1(GSP1) GCGAATCTCATCAGCA
      B386-2(GSP2) GGAAGCCGTAGTCAGAGG
      ORF C-F CTGACTACGGCTTCCAAAT
      C-R AACTGATCTTGCCATCTTCA
      qPCR q-1F TGCTGATGAGATTCGCCGACTTG
      q-1R TCGCTGAACTAACGGATTCTGCTG
      内参基因 internal genes 18sF TATACGCTAGTGGAGCTGGAA
      18sR GGGGAGGTAGTGACGAAAAAT

      Table 2.  The name and sequence of the primers

    • 基于测序验证无误的CN-B参考序列,并根据起始密码子和终止密码子确定其开放阅读框(ORF)的序列,通过RACE技术克隆来自中国明对虾肝胰腺和肌肉组织中CN-B的cDNA全长序列,使用Primer Premier 5.0软件设计3′ RACE和5′ RACE的特异性引物和巢式PCR反应的特异性引物(表2)。

    • 按照RevertAid First Strand cDNA Synthesis Kit试剂盒的说明书,采用巢式PCR,使用SUPERSCRIPT II RT酶和引物GSP-1(表2) 对总RNA进行CN-B第一链cDNA的合成,使用引物GSP-2(表2)对加了dC尾的cDNA进行PCR第一轮扩增。PCR反应程序:94 °C预变性2 min;94 °C变性30 s,55 °C退火30 s,72 °C延伸1 min,35个循环;72 °C最终延伸10 min。使用引物GSP-3(表2)和通用扩增引物AUAP进行巢式PCR第二轮扩增。

    • 使用逆转录酶SMARTScribe™ Reverse Transcriptase和引物3′RACE primer A对总RNA进行逆转录合成cDNA,使用引物C287-2和UPM (表2),以合成的3′ 端cDNA为模板进行第一轮PCR扩增。将第一轮PCR扩增产物稀释50倍,然后用引物C287-2.5和UPM(表2)进行第二轮PCR扩增。PCR反应程序按照5′RACE进行。

    • 将3′RACE和5′RACE第二轮扩增的PCR产物进行琼脂糖凝胶电泳检测,并对目的条带进行切胶回收和纯化,纯化产物连接到pMD18T,转化后对阳性克隆送交青岛派森诺基因生物技术有限公司进行测序,分别获得3′RACE和5′RACE的序列。利用BioEdit软件将克隆得到3′RACE、核心序列和5′RACE进行拼接获得CN-B的cDNA全长序列。

    • 利用NCBI中的BLAST主页程序进行主要的核苷酸序列以及氨基酸序列的比对。利用DNAMAN 8.0将CN-B的cDNA翻译成氨基酸序列;利用NetNGlyc 1.0 Server(http://www.cbs.dtu.dk/services/NetNGlyc/)进行糖基化位点分析;利用NetPhos 2.0 Serverr(http://www.cbs.dtu.dk/services/NetPhos/)进行磷酸化位点分析;利用ScanProsite tool(http://prosite.expasy.org/scanprosite/)进行二硫键分析;利用Protparam(http://web.expasy.org/compute_pi/)进行蛋白质等电点、分子量预测;利用ClustalW2(http://www.ebi.ac.uk/Tools/msa/clustalw2/)来进行氨酸序列的比对。基于BLAST搜索结果对calcineurin在氨基酸水平上做同源比较和进化树分析,使用DNAMAN 6.0软件进行同源多序列比对,使用SWISS-MODEL(http://swissmodel.expasy.org/)预测CN-B的蛋白质三维结构图;使用MEGA 6.0软件的邻接法(NJ)完成进化树构建,Bootstrap检测,重复1 000次。

    • 按照ChamQTM Universal SYBR qPCR Master Mix试剂盒(Vazyme)的说明书,通过Real-time PCR分析CN-B在HCG和LCG两组间5种组织中的表达谱,内参基因的引物18s-F和18s-R[22] 和目的基因引物q-1F和q-1R(表2)由生工生物工程(上海)股份有限公司合成。Real-time PCR在10 μL反应体系中进行:5 μLSYBR Master Mix主混合物,0.2 μLPrimer F和0.2 μLPrimer R,3.6 μL灭菌超纯水以及100 ng cDNA。反应条件为:95 °C初始变性30 s;95 °C变性5 s,55 °C退火34 s,40个循环;熔解曲线95 °C 15 s,55 °C 1 min,95 °C 15 s。使用2−△△CT方法[23]计算表达量;使用SPSS软件的Duncan法分析CN-B在HCG和LCG两组的同一组织中差异的显著性,若 P < 0.05,则认为差异显著;利用SigmaPlot 10.0软件完成相关图表的制作。

    2.   结果
    • 对前期转录组测序获得的CN-B的核心序列验证后,验证出其ORF序列的540 bp序列为正确的拼接结果。基于验证后的核心序列,通过RACE技术克隆获得了中国明对虾的CN-B的全长cDNA序列,命名为FcCN-B,GenBank登录号为:MK375257。FcCN-B的全长cDNA序列为2 867 bp,包括可以编码179个氨基酸的540 bp的ORF序列,95 bp的5′ UTR和2 232 bp的3′ UTR, 起始密码子是ATG,终止密码子是TAA。FcCN-B蛋白含有4个保守的EF-Hand钙离子结合结构域,分别为EF-1(aa 40~52),EF-2(aa 72~84),EF-3(aa 109~121)和EF-4(aa 150~162)(图1,下划线)。围绕 FcCN-B起始密码子处有Kozak共有序列(gccaatATGG)(图1,方框)。通过Protparam分析发现FcCN-B的理论分子量为20.34 ku,等电点为4.45。使用空间结构预测软件SWISS-MODEL,以Protein Data Bank中的1tco.1.B片段为模板进行FcCN-B的蛋白质三维结构预测,三级结构预测结果显示,FcCN-B蛋白包括α螺旋,β折叠片和EF-Hand钙离子结合结构域(图2,箭头所指处)。

      Figure 1.  Full-length of cDNA sequence and its corresponding amino sequence of FcCN-B gene

      Figure 2.  The three-dimensional structure prediction of FcCN-B protein

    • FcCN-B蛋白与无脊椎动物到哺乳动物对应的氨基酸序列都显示出高度保守的同一性。序列比对分析显示(图3),FcCN-B与人(Homo sapiens)、褐家鼠(Rattus norvegicus)、非洲爪蟾(Xenopus laevis)、黑腹果蝇(Drosophila melanogaster)、中华绒螯蟹、家蚕(Bombyx mori)、珠母贝(Pinctada fucata)和秀丽隐杆线虫(Caenorhabditis elegans)8种物种显示出较高(78.8%−93.8%)的序列同一性,分别为:人(84.9%)、褐家鼠(84.9%)、非洲爪蟾(84.3%)、黑腹果蝇(90.5%)、中华绒螯蟹(93.8%)、家蚕(88.2%)、珠母贝(86.0%)和秀丽隐杆线虫(78.8%),其中与中华绒螯蟹氨基酸序列的同源性最高,与秀丽隐杆线虫的同源性最低。

      Figure 3.  The alignment of FcCN-B amino acid sequence

    • FcCN-B基因编码的蛋白序列作系统进化分析结果显示,中国明对虾等无脊椎动物先与脊椎动物聚为两大分支,之后与较为低等的旋毛虫和秀丽隐杆线虫聚为一支。其中,中国明对虾与中华绒螯蟹紧密聚为一支,之后与黑腹果蝇和家蚕聚类关系最近(图4)。

      Figure 4.  The analysis of phylogenetic tree for CN-B

    • 在比较转录组分析结果中,与竞争能力弱组中的表达相比,FcCN-B在竞争能力强组中显著上调5.53倍(q value = 0.014)。在本研究中,采用Real-time PCR技术进一步分析FcCN-B在HCG和LCG两组间5种组织中的表达结果显示,在HCG组中,FcCN-B在神经节中的表达量最高,其次是心脏、肠、胃、肝胰腺;而在LCG中,FcCN-B在心脏中的表达量最高,其次是神经节、肠、胃、肝胰腺。FcCN-B在HCG组的神经节中的表达显著高于LCG组(P < 0.01);而在心脏中,FcCN-B在HCG组中的表达显著低于LCG组(P < 0.01)(图5)。结果提示该基因可能在竞争能力不同的对虾的神经节和心脏中发挥不同的作用。

      Figure 5.  Distribution of FcCN-B in five tissues between the HCG and LCG group of F. chinensis

    3.   讨论
    • 研究表明钙/钙调蛋白依赖性丝氨酸蛋白激酶基因(CASK)在果蝇的竞争行为中发挥重要的作用[24],而CASK参与的功能的实现需要在CN等重要基因的调节下共同完成[24-25]。我们推断CN-B可能参与中国明对虾的竞争行为过程,然而在对虾中还没有该基因的相关报道。因此,为了进一步明确CN-B在中国明对虾竞争行为中可能的作用,本研究通过RACE技术克隆了FcCN-B全长cDNA序列,并分析了其在竞争能力差异显著的两组间的不同组织中的表达情况,初步证明该基因在中国明对虾的竞争行为中可能发挥一定的作用,这在对虾中属于首次报道。

      CN是所有真核生物中,从寄生虫、线虫、果蝇、中华绒螯蟹等无脊椎动物到人类、褐家鼠等高等脊椎动物都表现出高度保守性的Ca2+ 结合蛋白。通过对FcCN-B编码区氨基酸序列的分析显示,FcCN-B蛋白与已报道的无脊椎动物和脊椎动物的CN氨基酸序列相比,都显示出非常高的序列同一性(约78%~94%)。FcCN-B蛋白同样含有4个保守的EF-Hand钙离子结合结构域,并且在果蝇、中华绒螯蟹等其他物种的CN-B蛋白中具有所有特征性保守结构域[21, 26],而且进化分析显示中国明对虾与中华绒螯蟹和黑腹果蝇的聚类关系最近,提示FcCN-B在中国明对虾中可能发挥与其在果蝇和中华绒螯蟹中相类似的功能。然而,聚类显示中国明对虾与旋毛虫和秀丽隐杆线虫的进化关系最远,这可能因为旋毛虫的寄生特点和秀丽隐杆线虫的雌雄同体特征导致CN-B在二者中的功能有别于其在中国明对虾中的作用。

      根据已有报道,CN在哺乳动物的中枢神经系统中最为丰富,具有极高的表达水平[27]。而且在水产动物中也有同样的发现,比如qRT-PCR定量分析结果显示中华绒螯蟹的EsCN-B主要在神经组织中表达[21],提示CN-B在神经组织中发挥更大的作用,这与本研究的结果基本一致。本研究结果显示,FcCN-B基因在中国明对虾的神经组织组中表达量较高。有意思的是,FcCN-B在中国明对虾竞争能力强组中的表达最高,且极显著高于其在竞争能力弱组中的表达,这与我们的比较转录组结果是一致的。结合已有的报道,即CASK基因参与果蝇的竞争过程,且CN基因可调节CASK发挥作用,FcCN-B在竞争能力强的中国明对虾的神经组织中的极高表达可能会使其对环境的变化(如饵料的出现和位置)做出更敏捷的反应,如此初步提示FcCN-B在中国明对虾的竞争行为中可能发挥一定的作用,这也有待于我们后续实验的进一步验证。其次,FcCN-B基因在中国明对虾的心脏组织也有较高的表达,而且其在竞争能力弱组中的表达显著高于竞争能力强组。已有研究显示,CN所在的钙调磷酸酶信号通路在肌纤维表型转化,结构重塑和细胞凋亡中起着至关重要的作用[17-20],且CN通路的组成部分参与心肌细胞肥大、左心室扩张的发展、心力衰竭和血管生成[28]。因此,结合以上研究结果,FcCN-B在中国明对虾竞争能力弱组中的显著高表达,可能会在一定程度上改变对虾心肌的功能降低了其对环境变化的反应能力。

      综上所述,本研究首次克隆得到了中国明对虾calcineurin B(FcCN-B)的cDNA全长序列,并通过Real-time PCR定量分析显示其在中国明对虾竞争能力显著不同的两组间的神经和心脏组织中的表达均表现出显著差异,结合已有的报道,初步证明该基因在中国明对虾的竞争行为中发挥一定的作用,本研究结果将为解析中国明对虾竞争行为的分子机制奠定重要的基础。

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