三疣梭子蟹蜕皮周期及眼柄切除后血淋巴甲基法尼酯浓度测定及其合成酶基因的表达分析
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摘要
甲基法尼酯(MF)是昆虫保幼激素III(JH III)的非环氧化物,是甲壳动物中JH的对等物,参与调控甲壳动物的各项生理活动。为了研究MF在蜕皮过程中的可能作用,利用气相色谱-质谱联用(GC-MS)中的选择离子监测模式(SIM)对三疣梭子蟹蜕皮周期中血淋巴中MF含量变化进行了检测,并利用实时荧光定量PCR(qPCR)对MF合成途径中关键酶的基因表达变化进行了分析。结果显示,三疣梭子蟹血淋巴MF含量在蜕皮前期显著上升,至D_1亚期升至最高。而AACT、FPS和JHAMT 3个MF合成酶在蜕皮周期中的基因表达模式虽然并不相同,但基本符合它们在MF合成通路中的位置关系,表明MF合成酶的转录调控可能存在级联效应。通过眼柄切除(ESA)进一步研究了眼柄因子对MF生物合成的调控作用,结果发现ESA能够迅速提升血淋巴MF含量和PtHMGR、PtFPS、PtFAMeT和PtJHAMT等基因的表达水平,这说明眼柄因子对MF的生物合成存在转录水平的调控。
Methyl farnesoate(MF)is the unepoxidized form of insect JH III, and is commonly considered as an equivalent of JH in crustaceans, which plays essential roles in many physiological processes of crustaceans. To investigate the possible role of MF in the molting process of the swimming crab, Portunus trituberculatus, the hemolymph levels were detected using the gas chromatography-mass spectrography(GC-MS)with selected ion monitoring(SIM), and the gene expression of the enzymes in MF biosynthesis pathway was detected using qPCR. The results showed that the hemolymph MF titer increased significantly in the pre-molt stage, and to the maximum in D_(1 )substage. The gene expression patterns of three MF biosynthesis enzymes including AACT, FPS, and JHAMT were not the same during the molt cycle, while still consistent with their location in the MF biosynthesis pathway, which indicated that the transcriptional regulation of MF synthesis enzymes might be cascaded. In addition, the regulatory role of eyestalk factor on the MF biosynthesis was investigated by eyestalk ablation(ESA)experiment. The results showed that ESA could rapidly induce the hemolymph MF titer and the gene expression of MF synthesis enzymes including PtHMGR, PtFPS, PtFAMeT and PtJHAMT, suggesting the existence of transcriptional regulation of eyestalk factors on MF biosynthesis.
Methyl farnesoate(MF)is the unepoxidized form of insect JH III, and is commonly considered as an equivalent of JH in crustaceans, which plays essential roles in many physiological processes of crustaceans. To investigate the possible role of MF in the molting process of the swimming crab, Portunus trituberculatus, the hemolymph levels were detected using the gas chromatography-mass spectrography(GC-MS)with selected ion monitoring(SIM), and the gene expression of the enzymes in MF biosynthesis pathway was detected using qPCR. The results showed that the hemolymph MF titer increased significantly in the pre-molt stage, and to the maximum in D_(1 )substage. The gene expression patterns of three MF biosynthesis enzymes including AACT, FPS, and JHAMT were not the same during the molt cycle, while still consistent with their location in the MF biosynthesis pathway, which indicated that the transcriptional regulation of MF synthesis enzymes might be cascaded. In addition, the regulatory role of eyestalk factor on the MF biosynthesis was investigated by eyestalk ablation(ESA)experiment. The results showed that ESA could rapidly induce the hemolymph MF titer and the gene expression of MF synthesis enzymes including PtHMGR, PtFPS, PtFAMeT and PtJHAMT, suggesting the existence of transcriptional regulation of eyestalk factors on MF biosynthesis.
引文
[1]ChANG E S, MYKLES D L. Regulation of crustacean molting: a review and our perspectives[J]. General & Comparative Endocrinology, 2011, 172(3):323-30.
[2]RIDDIFORD L M. How does juvenile hormone control insect metamorphosis and reproduction?[J]. General & Comparative Endocrinology, 2012, 179(3):477-484.
[3]NAGARAJU G P C. Is methyl farnesoate a crustacean hormone?[J]. Aquaculture, 2007, 272(14):39-54.
[4]LAUFER H, BORST D, BAKER F C, et al. Identification of a juvenile hormone-like compound in a crustacean.[J]. Science, 1987, 235(4785):202.
[5]LAUFER H, LANDAU M, HOMOLA E, et al. Methyl farnesoate: its site of synthesis and regulation of secretion in a juvenile crustacean[J]. Insect Biochemistry, 1987, 17(7):1129-1131.
[6]YUDIN A I, DIENER R A, CLARK W H, et al. Mandibular gland of the blue crab, Callinectes sapidus[J]. Biological Bulletin, 1980, 159(3):760-772.
[7]TAKETOMI T, MOTONO M, MIYAWAKI M. On the biological function of the mandibular gland of decapod crustacea[J]. Cell Biology International Reports, 1989, 13(5):463-469.
[8]ABDU U, BARKI A, KARPLUS I, et al. Physiological effects of methyl farnesoate and pyriproxyfen on wintering female crayfish Cherax quadricarinatus[J]. Aquaculture, 2001, 202(1):163-175.
[9]REDDY P R, NAGARAJU G P C, REDDY P S. Involvement of methyl farnesoate in the regulation of molting and reproduction in the freshwater crab oziotelphusa senex senex[J]. Journal of Crustacean Biology, 2004, 24(3):511-515.
[10]LAUFER H, DEMIR N, PAN X, et al. Methyl farnesoate controls adult male morphogenesis in the crayfish, Procambarus clarkii[J]. Journal of Insect Physiology, 2005, 51(4):379.
[11]HUI J H, TOBE S S, CHAN S M. Characterization of the putative farnesoic acid O-methyltransferase (LvFAMeT)cDNA from white shrimp, Litopenaeus vannamei evidence for its role in molting[J]. Peptides, 2008, 29(2):252-260.
[12]TAMONE S L, CHANG E S. Methyl farnesoate stimulates ecdysteroid secretion from crab Y-organs in vitro[J]. General & Comparative Endocrinology, 1993, 89(3):425-432.
[13]GHANAWI J, SAOUD I P. Molting, reproductive biology, and hatchery management of red claw crayfish Cherax quadricarinatus (von Martens 1868)[J]. Aquaculture, 2012, s358 359(1):183-195.
[14]BYARD E H, SHIVERS R R, AIKEN D E. The mandibular organ of the lobster, Homarus americanus[J]. Cell & Tissue Research, 1975, 162(1):13-22.
[15]FELTERMAN M, ZOU E. The exogenous methyl farnesoate does not impact ecdysteroid signaling in the crustacean epidermis in vivo[J]. Aquaculture, 2011, 317(14):251-254.
[16]BELL S X, MART N D, PIULACHS M D. The mevalonate pathway and the synthesis of juvenile hormone in insects[J]. Annual Review of Entomology, 2005, 50(1):181.
[17]HUI J H L, HAYWARD A, BENDENA W G, et al. Evolution and functional divergence of enzymes involved in sesquiterpenoid hormone biosynthesis in crustaceans and insects[J]. Peptides, 2010, 31(3):451-455.
[18]邱锡尔, 朱冬发, 汤洁,等. 甲基法尼酯在甲壳动物中的生理作用及其机制的研究进展[J]. 生物学杂志, 2015(1):76-81.
[19]SIN Y W, KENNY N J, QU Z, et al. Identification of putative ecdysteroid and juvenile hormone pathway genes in the shrimp Neocaridina denticulata[J]. General & Comparative Endocrinology, 2015, 214:167.
[20]XIE X, TAO T, LIU M, et al. The potential role of juvenile hormone acid methyltransferase in methyl farnesoate (MF)biosynthesis in the swimming crab, Portunus trituberculatus[J]. Animal Reproduction Science, 2016, 168:40-49.
[21]VENTURA T, MANOR R, AFLALO E D, et al. Post-embryonic transcriptomes of the prawn Macrobrachium rosenbergii: multigenic succession through metamorphosis[J]. PLoS One, 2013, 8(1): e55322.
[22]LI S, FRIESEN J A, HOLFORD K C, et al. Methyl farnesoate synthesis in the lobster mandibular organ: the roles of HMG-CoA reductase and farnesoic acid O-methyltransferase[J]. Comparative Biochemistry & Physiology Part A Molecular & Integrative Physiology, 2010, 155(1):49-55.
[23]王凤皎, 朱冬发, 邱锡尔,等. 三疣梭子蟹AACT基因克隆及其在卵巢发育中的表达分析[J]. 水产学报, 2015, 39(6):790-798.
[24]朱韬, 朱冬发, 邱锡尔,等. 三疣梭子蟹fps克隆及其在卵巢发育中的表达分析[J]. 生物学杂志, 2016, 33(3):10-14.
[25]沈洁, 朱冬发, 胡则辉,等. 三疣梭子蟹蜕皮周期的分期[J]. 水产学报, 2011, 35(10):1481-1487.
[26]XI X, ZHU D, YAN L, et al. Hemolymph levels of methyl farnesoate during ovarian development of the swimming crab Portunus trituberculatus, and its relation to transcript levels of HMG-CoA reductase and farnesoic acid O-methyltransferase[J]. Biological Bulletin, 2015, 228(2):118.
[27]WILDER M N, OKADA S, FUSETANI N, et al. Hemolymph profiles of juvenoid substances in the giant freshwater prawn Macrobrachium rosenbergii in relation to reproduction and molting[J]. Fisheries Science, 2008, 61, 175-176.
[28]汪春建, 朱冬发, 亓一舟,等. 三疣梭子蟹蜕皮周期中MIH基因mRNA水平与蜕皮激素浓度变化[J]. 水生生物学报, 2013, 37(1):22-28.
[29]邱锡尔, 朱冬发, 崔晓雨,等. 三疣梭子蟹HMGR基因的克隆及其在蜕皮中的表达分析[J]. 海洋与湖沼, 2014, 45(6):1192-1201.
[30]谢熙, 朱冬发, 崔晓雨,等. 三疣梭子蟹FAMeT基因克隆及其在蜕皮周期中的表达水平[J]. 水产学报, 2013, 37(7):994-1001.
[31]LIU L, LAUFER H. Isolation and characterization of sinus gland neuropeptides with both mandibular organ inhibiting and hyperglycemic effects from the spider crab Libinia emarginata[J]. Archives of Insect Biochemistry & Physiology, 1996, 32(3/4):375-385.
[32]WAINWRIGHT G, WEBSTER S G, WILKINSON M C, et al. Structure and significance of mandibular organ-inhibiting hormone in the crab, cancer pagurus involvement in multihormonal regulation of growth and reproduction[J]. Journal of Biological Chemistry, 1996, 271(22): 12749-12754.
[2]RIDDIFORD L M. How does juvenile hormone control insect metamorphosis and reproduction?[J]. General & Comparative Endocrinology, 2012, 179(3):477-484.
[3]NAGARAJU G P C. Is methyl farnesoate a crustacean hormone?[J]. Aquaculture, 2007, 272(14):39-54.
[4]LAUFER H, BORST D, BAKER F C, et al. Identification of a juvenile hormone-like compound in a crustacean.[J]. Science, 1987, 235(4785):202.
[5]LAUFER H, LANDAU M, HOMOLA E, et al. Methyl farnesoate: its site of synthesis and regulation of secretion in a juvenile crustacean[J]. Insect Biochemistry, 1987, 17(7):1129-1131.
[6]YUDIN A I, DIENER R A, CLARK W H, et al. Mandibular gland of the blue crab, Callinectes sapidus[J]. Biological Bulletin, 1980, 159(3):760-772.
[7]TAKETOMI T, MOTONO M, MIYAWAKI M. On the biological function of the mandibular gland of decapod crustacea[J]. Cell Biology International Reports, 1989, 13(5):463-469.
[8]ABDU U, BARKI A, KARPLUS I, et al. Physiological effects of methyl farnesoate and pyriproxyfen on wintering female crayfish Cherax quadricarinatus[J]. Aquaculture, 2001, 202(1):163-175.
[9]REDDY P R, NAGARAJU G P C, REDDY P S. Involvement of methyl farnesoate in the regulation of molting and reproduction in the freshwater crab oziotelphusa senex senex[J]. Journal of Crustacean Biology, 2004, 24(3):511-515.
[10]LAUFER H, DEMIR N, PAN X, et al. Methyl farnesoate controls adult male morphogenesis in the crayfish, Procambarus clarkii[J]. Journal of Insect Physiology, 2005, 51(4):379.
[11]HUI J H, TOBE S S, CHAN S M. Characterization of the putative farnesoic acid O-methyltransferase (LvFAMeT)cDNA from white shrimp, Litopenaeus vannamei evidence for its role in molting[J]. Peptides, 2008, 29(2):252-260.
[12]TAMONE S L, CHANG E S. Methyl farnesoate stimulates ecdysteroid secretion from crab Y-organs in vitro[J]. General & Comparative Endocrinology, 1993, 89(3):425-432.
[13]GHANAWI J, SAOUD I P. Molting, reproductive biology, and hatchery management of red claw crayfish Cherax quadricarinatus (von Martens 1868)[J]. Aquaculture, 2012, s358 359(1):183-195.
[14]BYARD E H, SHIVERS R R, AIKEN D E. The mandibular organ of the lobster, Homarus americanus[J]. Cell & Tissue Research, 1975, 162(1):13-22.
[15]FELTERMAN M, ZOU E. The exogenous methyl farnesoate does not impact ecdysteroid signaling in the crustacean epidermis in vivo[J]. Aquaculture, 2011, 317(14):251-254.
[16]BELL S X, MART N D, PIULACHS M D. The mevalonate pathway and the synthesis of juvenile hormone in insects[J]. Annual Review of Entomology, 2005, 50(1):181.
[17]HUI J H L, HAYWARD A, BENDENA W G, et al. Evolution and functional divergence of enzymes involved in sesquiterpenoid hormone biosynthesis in crustaceans and insects[J]. Peptides, 2010, 31(3):451-455.
[18]邱锡尔, 朱冬发, 汤洁,等. 甲基法尼酯在甲壳动物中的生理作用及其机制的研究进展[J]. 生物学杂志, 2015(1):76-81.
[19]SIN Y W, KENNY N J, QU Z, et al. Identification of putative ecdysteroid and juvenile hormone pathway genes in the shrimp Neocaridina denticulata[J]. General & Comparative Endocrinology, 2015, 214:167.
[20]XIE X, TAO T, LIU M, et al. The potential role of juvenile hormone acid methyltransferase in methyl farnesoate (MF)biosynthesis in the swimming crab, Portunus trituberculatus[J]. Animal Reproduction Science, 2016, 168:40-49.
[21]VENTURA T, MANOR R, AFLALO E D, et al. Post-embryonic transcriptomes of the prawn Macrobrachium rosenbergii: multigenic succession through metamorphosis[J]. PLoS One, 2013, 8(1): e55322.
[22]LI S, FRIESEN J A, HOLFORD K C, et al. Methyl farnesoate synthesis in the lobster mandibular organ: the roles of HMG-CoA reductase and farnesoic acid O-methyltransferase[J]. Comparative Biochemistry & Physiology Part A Molecular & Integrative Physiology, 2010, 155(1):49-55.
[23]王凤皎, 朱冬发, 邱锡尔,等. 三疣梭子蟹AACT基因克隆及其在卵巢发育中的表达分析[J]. 水产学报, 2015, 39(6):790-798.
[24]朱韬, 朱冬发, 邱锡尔,等. 三疣梭子蟹fps克隆及其在卵巢发育中的表达分析[J]. 生物学杂志, 2016, 33(3):10-14.
[25]沈洁, 朱冬发, 胡则辉,等. 三疣梭子蟹蜕皮周期的分期[J]. 水产学报, 2011, 35(10):1481-1487.
[26]XI X, ZHU D, YAN L, et al. Hemolymph levels of methyl farnesoate during ovarian development of the swimming crab Portunus trituberculatus, and its relation to transcript levels of HMG-CoA reductase and farnesoic acid O-methyltransferase[J]. Biological Bulletin, 2015, 228(2):118.
[27]WILDER M N, OKADA S, FUSETANI N, et al. Hemolymph profiles of juvenoid substances in the giant freshwater prawn Macrobrachium rosenbergii in relation to reproduction and molting[J]. Fisheries Science, 2008, 61, 175-176.
[28]汪春建, 朱冬发, 亓一舟,等. 三疣梭子蟹蜕皮周期中MIH基因mRNA水平与蜕皮激素浓度变化[J]. 水生生物学报, 2013, 37(1):22-28.
[29]邱锡尔, 朱冬发, 崔晓雨,等. 三疣梭子蟹HMGR基因的克隆及其在蜕皮中的表达分析[J]. 海洋与湖沼, 2014, 45(6):1192-1201.
[30]谢熙, 朱冬发, 崔晓雨,等. 三疣梭子蟹FAMeT基因克隆及其在蜕皮周期中的表达水平[J]. 水产学报, 2013, 37(7):994-1001.
[31]LIU L, LAUFER H. Isolation and characterization of sinus gland neuropeptides with both mandibular organ inhibiting and hyperglycemic effects from the spider crab Libinia emarginata[J]. Archives of Insect Biochemistry & Physiology, 1996, 32(3/4):375-385.
[32]WAINWRIGHT G, WEBSTER S G, WILKINSON M C, et al. Structure and significance of mandibular organ-inhibiting hormone in the crab, cancer pagurus involvement in multihormonal regulation of growth and reproduction[J]. Journal of Biological Chemistry, 1996, 271(22): 12749-12754.