三疣梭子蟹PtDNMT1基因的克隆及其在低盐适应中的表达分析
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摘要
甲基转移酶是维持基因组甲基化状态的重要基因,本研究利用SMART-RACE技术克隆了三疣梭子蟹(Portunus trituberculatus)甲基转移酶基因(PtDNMT1)。PtDNMT1基因cDNA序列全长5919 bp,包括4832 bp的开放阅读框,编码1610个氨基酸,预测分子量为148.15 kDa,理论等电点为4.68。结构预测发现,Pt DNMT1有2个特殊的结构域,分别是锌指结构域(zf-CXXC)和甲基转移酶家族特有的Dcm结构域。进化树分析显示,PtDNMT1基因与昆虫类的DNMT基因聚为一支。组织表达分析发现,PtDNMT1基因在肝胰腺、鳃、卵巢、肌肉、胃、心脏、血液中均有表达,其中在肝胰腺中表达最高,卵巢和鳃次之。进一步研究了低盐胁迫后PtDNMT1基因在鳃、肝胰腺和肌肉组织中的表达变化规律为胁迫6 h时鳃组织中PtDNMT1基因的表达即达到峰值(5.3倍),并一直持续到12 h (4倍),随后逐步下降,在72 h时仍显著高于对照组(2.3倍);PtDNMT1基因在肝胰腺中的表达规律类似于鳃,然而其达到峰值的时间稍晚于鳃(24 h),且上调倍数高于鳃(8倍);低盐胁迫后Pt DNMT1基因在肌肉中的表达最初呈现下调趋势,之后(24h)上调表达至峰值(2.2倍),且一直上调表达至72 h。本研究首次克隆了PtDNMT1基因,根据其在各组织中的表达分布特征以及盐度胁迫后的表达变化情况,推测DNA甲基化在三疣梭子蟹低盐适应中发挥了重要作用。
Methyltransferase(DNMT1) is an important gene that maintains the methylation state of the genome. This study uses the SMART-RACE technique to clone the DNMT1 gene of Portunus trituberculatus(PtDNMT1). The cDNA sequence of PtDNMT1 gene is 5919 bp, including the open reading frame of 4832 bp, and 1610 amino acids are encoded. The predicted molecular weight is 148.15 kDa, and the theoretical isoelectric point is 4.68. The structure prediction found that there are two special PtDNMT1 domain structures: the zf-CXXC zinc finger domain and the methyltransferase familial Dcm structure domain. The analysis of the evolutionary tree showed that PtDNMT1 gene and insects DNMTgather into one. PtDNMT1 was expressed in the hepatopancreas, gill, ovary, muscle, stomach, heart, and blood. The highest expression was found in the hepatopancreas, followed by the ovaries and gills. Then we get a further research about the PtDNMT1 gene expression during low salinity stress in gill, l hepatopancreas and muscle tissue. In gill tissue, we found that Pt DNMT1 gene expression peaked(5 folds) after 6 h, and continued to 12 h(6 folds), then declined gradually, but was still significantly higher than the control group(3 folds). The expression of Pt DNMT1 in the hepatopancreas was similar to the gills, however the peak time was later than in gill tissue(24 h), and the peak was higher than in the gills(8 folds). After low salinity stress, the expression of the PtDNMT1 gene in muscle was firstly reduced, then(24 h) increased to a peak(2.2 times), and the expression was raised to 72 h. This study is the first to clone the Pt DNMT1 gene. According to its distribution characteristics and occurrence in various organizations, expression changes under salinity stress. We speculate that low salinity adaptations in DNA methylation in P. trituberculatus played an important role.
Methyltransferase(DNMT1) is an important gene that maintains the methylation state of the genome. This study uses the SMART-RACE technique to clone the DNMT1 gene of Portunus trituberculatus(PtDNMT1). The cDNA sequence of PtDNMT1 gene is 5919 bp, including the open reading frame of 4832 bp, and 1610 amino acids are encoded. The predicted molecular weight is 148.15 kDa, and the theoretical isoelectric point is 4.68. The structure prediction found that there are two special PtDNMT1 domain structures: the zf-CXXC zinc finger domain and the methyltransferase familial Dcm structure domain. The analysis of the evolutionary tree showed that PtDNMT1 gene and insects DNMTgather into one. PtDNMT1 was expressed in the hepatopancreas, gill, ovary, muscle, stomach, heart, and blood. The highest expression was found in the hepatopancreas, followed by the ovaries and gills. Then we get a further research about the PtDNMT1 gene expression during low salinity stress in gill, l hepatopancreas and muscle tissue. In gill tissue, we found that Pt DNMT1 gene expression peaked(5 folds) after 6 h, and continued to 12 h(6 folds), then declined gradually, but was still significantly higher than the control group(3 folds). The expression of Pt DNMT1 in the hepatopancreas was similar to the gills, however the peak time was later than in gill tissue(24 h), and the peak was higher than in the gills(8 folds). After low salinity stress, the expression of the PtDNMT1 gene in muscle was firstly reduced, then(24 h) increased to a peak(2.2 times), and the expression was raised to 72 h. This study is the first to clone the Pt DNMT1 gene. According to its distribution characteristics and occurrence in various organizations, expression changes under salinity stress. We speculate that low salinity adaptations in DNA methylation in P. trituberculatus played an important role.
引文
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Huang WY,Zhang HJ,Xing YX,et al.Effects of NaCl stress on seed germination and DNA methylation status detected by MSAP analysis in cucumber.Scientia Agricultura Sinica,2013,46(8):1646-1656[黄韫宇,张海军,邢燕霞,等.NaCl胁迫对黄瓜种子萌发的影响及DNA甲基化的MSAP分析.中国农业科学,2013,46(8):1646-1656]
Jiang S,Xu QH.Influence of salinity stress on the activity of gill Na+/K+-ATPase in swimming crab(Portunus trituberculatus).Journal of Fisheries of China,2011,35(10):1475-1480[江山,许强华.盐度胁迫对三疣梭子蟹鳃Na+/K+-ATPase活力的影响.水产学报,2011,35(10):1475-1480]
Li N,Zhang Y,Xie LN,et al.Research progress in DNAmethylation in plants.Plant Physiology Journal,2012,48(11):1027-1036[李娜,张旸,解莉楠,等.植物DNA甲基化研究进展.植物生理学报,2012,48(11):1027-1036]
Lu YL,Wang F,Zhao ZY,et al.Effects of salinity on growth,molt and energy utilization of juvenile swimming crab Portunus trituberculatus.Journal of Fishery Sciences of China,2012,19(2):237-245[路允良,王芳,赵卓英,等.盐度对三疣梭子蟹生长、蜕壳及能量利用的影响.中国水产科学,2012;19(2):237-245]
LüF,Huang JT,Yu YB,et al.Effects of salinity on growth,muscle composition,and protease activity of Portunus trituberculatus.Transactions of Oceanology and Limnology,2010(4):137-142[吕富,黄金田,於叶兵,等.盐度对三疣梭子蟹生长、肌肉组成及蛋白酶活性的影响.海洋湖沼通报,2010(4):137-142]
LüJ,Liu P,Wang Y,et al.Transcriptome analysis of Portunus trituberculatus in response to salinity stress provides insights into the molecular basis of osmoregulation.PLo SOne,2013,8(12):e82155
Ma JW,LüJJ,Liu P,et al.Na+/H+-exchanger in swimming crab(Portunus trituberculatus):Cloning,characterization and m RNA expression under salinity stress.Acta Hydrobiologica Sinica,2016,40(5):902-906[马金武.吕建建,刘萍,等.三疣梭子蟹Na+/H+-exchanger基因克隆鉴定及在盐度胁迫下的表达分析.水生生物学报,2016,40(5):902-907]
Rosa R,Nunes M.Biochemical changes during the reproductive cycle of the deep-sea decapod Nephrops norvegicus on the south coast of Portugal.Marine Biology,2002,141(6):1001-1009
Sui YM,Gao BQ,Liu P,et al.The tolerance to and optimal salinity for growth in swimming crab Portunus trituberculatus“Huangxuan No.1”.Journal of Dalian Fisheries University,2012,27(5):398-401[隋延鸣,高保全,刘萍,等.三疣梭子蟹“黄选1号”盐度耐受性及适宜生长盐度分析.大连海洋大学学报,2012,27(5):398-401]
Sui YM,Gao BQ,Liu P,et al.Preliminary studies on the salinity tolerance of“Huangxuan No.1”Portunus trituberculatus.Progress in Fishery Sciences,2012,33(2):63-68[隋延鸣,高保全,刘萍,等.三疣梭子蟹“黄选1号”盐度耐受性分析.渔业科学进展,2012,33(2):63-68]
Tan Y,Xu QH.The prokaryotic expression of recombinant heat shock protein HSP90a of Portunus trituberculatus under salinity stress.Journal of Fisheries of China,2012,36(5):681-684[覃烨,许强华.盐度胁迫下三疣梭子蟹热休克蛋白HSP90a的原核表达.水产学报,2012,36(5):681-684]
Wang C,Jiang LX,Wang RJ,et al.Effect of abrupt and gradual changes in salinity on development and feeding in juvenile swimming crab(Portunus trituberculatus).Fisheries Science,2010,29(9):510-514[王冲,姜令绪,王仁杰,等.盐度骤变和渐变对三疣梭子蟹幼蟹发育和摄食的影响.水产科学,2010,29(9):510-514]
Wang Y,LüJJ,Liu P,et al.Cloning and characterization of aquaporins 1 and its expression analysis under salinity stress in Portunus trituberculatus.Journal of Fishery Sciences of China,2014,21(5):893-901[王渝,吕建建,刘萍,等.三疣梭子蟹水通道蛋白1 c DNA及其盐度胁迫下的表达分析.中国水产科学,2014,21(5):893-901]
Wang Y,LüJJ,Liu P,et al.Cloning and characterization of chloride intracellular channel gene and its expression under low salinity stress in Portunus trituberculatus.Oceanologia et Limnologia Sinica,2014,45(6):1359-1366[王瑜,吕建建,刘萍,等.三疣梭子蟹胞内氯离子通道蛋白基因克隆及其表达分析.海洋与湖沼,2014,45(6):1359-1366]
Wu B,Yang AG.MSAP analysis on genome-wide DNAmethylation in selected and wild Japanese scallop Patinopecten yessoensis.Oceanologla et Limnologla Sinica,2015,46(4):976-981[吴彪,杨爱国.虾夷扇贝选育群体与野生群体基因组DNA甲基化的MSAP分析.海洋与湖沼,2015,46(4):976-982]
Yang XD,Han W,Liu F,et al.DNA methylation in vertebrate embryogenesis.Hereditas,2012,34(9):1108-1113[杨晓丹,韩威,刘峰,等.DNA甲基化与脊椎动物胚胎发育.遗传,2012,34(9):1108-1113]
Zhang L,Xie BH,Zhang QY,et al.The cloning and expression analysis of the DNMT1 gene cDNA of Crucian carp.Acta Hydrobiologica Sinica.Journal of Fisheries of China,2010,34(2):230-234[张蕾,谢冰花,张琼宇,等.鲫鱼DNMT1基因c DNA的克隆及表达分析.水产学报,2010,34(2):229-235]
Zheng PP,Wang CL,Song WW,et al.Effect of salinity stress on serum non-specific immune factors in swimming crab Portunus trituberculatus.Fisheries Science,2010,29(11):634-638[郑萍萍,王春琳,宋微微,等.盐度胁迫对三疣梭子蟹血清非特异性免疫因子的影响.水产科学,2010,29(11):634-638]
Zhou CL,Yin YF,Zhang J,et al.Biological implications of DNAmethylation and DNA methylation assays.Journal of Nanhua University(Medical Edition),2005,33(2):148-152[周翠兰,殷宇芳,张佳,等.DNA甲基化的生物学意义及其检测方法.南华大学学报(医学版),2005,33(2):148-153]
Zhu HP,Lu MX,Huang ZH,et al.Effect of low temperature on genomic DNA methylation in Nile tilapia(Oreochromis niloticus).Journal of Fisheries of China,2013,31(10):1460-1467[朱华平,卢迈新,黄樟翰,等.低温对罗非鱼基因组DNA甲基化的影响.水产学报,2013,37(10):1460-1467]
Fu P,LüJJ,Liu P,et al.Effects of different salinity level on free amino acid composition in muscle and hemolymph of the swimming crab Portunus trituberculatus.Journal of Fisheries of China,2017,41(3):374-381[付萍,吕建建,刘萍,等.盐度胁迫对三疣梭子蟹肌肉和血淋巴中游离氨基酸含量的影响.水产学报,2017,41(3):374-381]
He MC,Zhang JY,Liu CL,et al.DNA methylation level and pattern in growing sepia esculenta hoyle.Progress in Fishery Sciences,2018,39(4):46-55[何暮春,张金勇,刘长琳,等.金乌贼生长期DNA甲基化水平和模式.渔业科学进展,2018,39(4):46-55]
Huang WY,Zhang HJ,Xing YX,et al.Effects of NaCl stress on seed germination and DNA methylation status detected by MSAP analysis in cucumber.Scientia Agricultura Sinica,2013,46(8):1646-1656[黄韫宇,张海军,邢燕霞,等.NaCl胁迫对黄瓜种子萌发的影响及DNA甲基化的MSAP分析.中国农业科学,2013,46(8):1646-1656]
Jiang S,Xu QH.Influence of salinity stress on the activity of gill Na+/K+-ATPase in swimming crab(Portunus trituberculatus).Journal of Fisheries of China,2011,35(10):1475-1480[江山,许强华.盐度胁迫对三疣梭子蟹鳃Na+/K+-ATPase活力的影响.水产学报,2011,35(10):1475-1480]
Li N,Zhang Y,Xie LN,et al.Research progress in DNAmethylation in plants.Plant Physiology Journal,2012,48(11):1027-1036[李娜,张旸,解莉楠,等.植物DNA甲基化研究进展.植物生理学报,2012,48(11):1027-1036]
Lu YL,Wang F,Zhao ZY,et al.Effects of salinity on growth,molt and energy utilization of juvenile swimming crab Portunus trituberculatus.Journal of Fishery Sciences of China,2012,19(2):237-245[路允良,王芳,赵卓英,等.盐度对三疣梭子蟹生长、蜕壳及能量利用的影响.中国水产科学,2012;19(2):237-245]
LüF,Huang JT,Yu YB,et al.Effects of salinity on growth,muscle composition,and protease activity of Portunus trituberculatus.Transactions of Oceanology and Limnology,2010(4):137-142[吕富,黄金田,於叶兵,等.盐度对三疣梭子蟹生长、肌肉组成及蛋白酶活性的影响.海洋湖沼通报,2010(4):137-142]
LüJ,Liu P,Wang Y,et al.Transcriptome analysis of Portunus trituberculatus in response to salinity stress provides insights into the molecular basis of osmoregulation.PLo SOne,2013,8(12):e82155
Ma JW,LüJJ,Liu P,et al.Na+/H+-exchanger in swimming crab(Portunus trituberculatus):Cloning,characterization and m RNA expression under salinity stress.Acta Hydrobiologica Sinica,2016,40(5):902-906[马金武.吕建建,刘萍,等.三疣梭子蟹Na+/H+-exchanger基因克隆鉴定及在盐度胁迫下的表达分析.水生生物学报,2016,40(5):902-907]
Rosa R,Nunes M.Biochemical changes during the reproductive cycle of the deep-sea decapod Nephrops norvegicus on the south coast of Portugal.Marine Biology,2002,141(6):1001-1009
Sui YM,Gao BQ,Liu P,et al.The tolerance to and optimal salinity for growth in swimming crab Portunus trituberculatus“Huangxuan No.1”.Journal of Dalian Fisheries University,2012,27(5):398-401[隋延鸣,高保全,刘萍,等.三疣梭子蟹“黄选1号”盐度耐受性及适宜生长盐度分析.大连海洋大学学报,2012,27(5):398-401]
Sui YM,Gao BQ,Liu P,et al.Preliminary studies on the salinity tolerance of“Huangxuan No.1”Portunus trituberculatus.Progress in Fishery Sciences,2012,33(2):63-68[隋延鸣,高保全,刘萍,等.三疣梭子蟹“黄选1号”盐度耐受性分析.渔业科学进展,2012,33(2):63-68]
Tan Y,Xu QH.The prokaryotic expression of recombinant heat shock protein HSP90a of Portunus trituberculatus under salinity stress.Journal of Fisheries of China,2012,36(5):681-684[覃烨,许强华.盐度胁迫下三疣梭子蟹热休克蛋白HSP90a的原核表达.水产学报,2012,36(5):681-684]
Wang C,Jiang LX,Wang RJ,et al.Effect of abrupt and gradual changes in salinity on development and feeding in juvenile swimming crab(Portunus trituberculatus).Fisheries Science,2010,29(9):510-514[王冲,姜令绪,王仁杰,等.盐度骤变和渐变对三疣梭子蟹幼蟹发育和摄食的影响.水产科学,2010,29(9):510-514]
Wang Y,LüJJ,Liu P,et al.Cloning and characterization of aquaporins 1 and its expression analysis under salinity stress in Portunus trituberculatus.Journal of Fishery Sciences of China,2014,21(5):893-901[王渝,吕建建,刘萍,等.三疣梭子蟹水通道蛋白1 c DNA及其盐度胁迫下的表达分析.中国水产科学,2014,21(5):893-901]
Wang Y,LüJJ,Liu P,et al.Cloning and characterization of chloride intracellular channel gene and its expression under low salinity stress in Portunus trituberculatus.Oceanologia et Limnologia Sinica,2014,45(6):1359-1366[王瑜,吕建建,刘萍,等.三疣梭子蟹胞内氯离子通道蛋白基因克隆及其表达分析.海洋与湖沼,2014,45(6):1359-1366]
Wu B,Yang AG.MSAP analysis on genome-wide DNAmethylation in selected and wild Japanese scallop Patinopecten yessoensis.Oceanologla et Limnologla Sinica,2015,46(4):976-981[吴彪,杨爱国.虾夷扇贝选育群体与野生群体基因组DNA甲基化的MSAP分析.海洋与湖沼,2015,46(4):976-982]
Yang XD,Han W,Liu F,et al.DNA methylation in vertebrate embryogenesis.Hereditas,2012,34(9):1108-1113[杨晓丹,韩威,刘峰,等.DNA甲基化与脊椎动物胚胎发育.遗传,2012,34(9):1108-1113]
Zhang L,Xie BH,Zhang QY,et al.The cloning and expression analysis of the DNMT1 gene cDNA of Crucian carp.Acta Hydrobiologica Sinica.Journal of Fisheries of China,2010,34(2):230-234[张蕾,谢冰花,张琼宇,等.鲫鱼DNMT1基因c DNA的克隆及表达分析.水产学报,2010,34(2):229-235]
Zheng PP,Wang CL,Song WW,et al.Effect of salinity stress on serum non-specific immune factors in swimming crab Portunus trituberculatus.Fisheries Science,2010,29(11):634-638[郑萍萍,王春琳,宋微微,等.盐度胁迫对三疣梭子蟹血清非特异性免疫因子的影响.水产科学,2010,29(11):634-638]
Zhou CL,Yin YF,Zhang J,et al.Biological implications of DNAmethylation and DNA methylation assays.Journal of Nanhua University(Medical Edition),2005,33(2):148-152[周翠兰,殷宇芳,张佳,等.DNA甲基化的生物学意义及其检测方法.南华大学学报(医学版),2005,33(2):148-153]
Zhu HP,Lu MX,Huang ZH,et al.Effect of low temperature on genomic DNA methylation in Nile tilapia(Oreochromis niloticus).Journal of Fisheries of China,2013,31(10):1460-1467[朱华平,卢迈新,黄樟翰,等.低温对罗非鱼基因组DNA甲基化的影响.水产学报,2013,37(10):1460-1467]