黄条鰤PTEN基因克隆、组织分布及早期发育阶段的表达分析
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摘要
为探究PTEN在黄条鰤Seriola aureovittata生长发育过程中的分子作用机制,采用同源克隆和cDNA末端快速扩增(RACE)技术克隆了黄条鰤生长调控因子PTEN的cDNA全长序列,并采用实时荧光定量PCR(qPCR)技术分析了PTEN mRNA的时空表达特征。结果表明:黄条鰤PTEN cDNA序列全长为1575 bp,开放阅读框为1287 bp,编码428个氨基酸,PTEN编码的蛋白具有脊椎动物PTEN的共同特征,含有PTP(PTPc基序)和PTEN-C2结构域;序列比对分析发现,黄条鰤与高体鰤Seriola dumerili同源性较高,一致性达到了95%;通过系统进化树分析表明,黄条鰤与鲈形目及鲽形目鱼类聚为一支;经qPCR分析发现,黄条鰤PTEN mRNA在心脏中的相对表达量最高(P<0.05),其次在脾脏、头肾、肾脏、胃、肠、鳃中表达量较高,PTEN mRNA在多种组织中的广泛表达,暗示着其可能参与多种生理过程的调控;同时分析了PTEN mRNA在黄条鰤早期发育阶段(胚胎期、仔稚幼鱼期)中的时序表达特征,发现PTEN在早期胚胎发育中持续表达,从低囊胚时期(38 hpf)表达量显著升高(P<0.05),初孵仔鱼时期相对表达量最高(P<0.05);在仔稚幼鱼时期,PTEN mRNA相对表达水平逐渐降低,35日龄表达量显著下降(P<0.05)并保持较低表达水平。研究表明,PTEN在胚胎及仔稚幼鱼发育过程中具有重要调节作用,本研究结果可为黄条鰤早期生长发育过程的调控机制研究及苗种培育提供数据资料。
The full-length cDNA sequence of the growth regulator PTEN was cloned by homologous cloning and rapid amplification of cDNA ends(RACE),and temporal and spatial expression of PTEN mRNA was analyzed in goldstriped amberjack Seriola aureovittata by quantitative PCR(qPCR) to explore the molecular mechanism of PTEN in the growth and development of goldstriped amberjack. The results showed that the PTEN cDNA sequence was 1575 bp in length and the ORF was 1287 bp, encoding 428 amino acids. PTEN has the common feature of vertebrate PTEN and contains PTP(PTPc motif) and PTEN-C2 domain. Sequence alignment analysis revealed that both goldstriped amberjack and greater amberjack Seriola dumerili had a high homology(95%). The phylogenetic tree indicated that goldstriped amberjack PTEN was clustered into one branch with the PTEN Perciformes and Pleuronectiformes.By qPCR analysis, the maximal relative expression of PTEN was observed in the heart of goldstriped amberjack(P<0.05), followed by in the spleen, head kidney, kidney, stomach, intestine, gill and pituitary, indicating that the PTEN is involved in the regulation of a variety of physiological processes in various tissues due to high expression of PTEN mRNA. It was found that PTEN was continuously expressed in early embryonic development(embryonic stage, larvae and juvenile), with significantly higher expression level from early blastula stage(38 hours post fertilization, hpf)(P<0.05) with the maximal expression level in freshly hatched larvae(P<0.05). The expression level of PTEN mRNA was gradually decreased during the postlarvae and juvenile(P<0.05). The expression level of PTEN was found to be gradually decreased, significantly and constantly low at 35 days old. The findings indicate that PTEN plays an important regulatory role in the development of embryos and juveniles, which provides a theoretical basis with research of the regulation mechanism and culture during the early growth and development stages in goldstriped amberjack.
The full-length cDNA sequence of the growth regulator PTEN was cloned by homologous cloning and rapid amplification of cDNA ends(RACE),and temporal and spatial expression of PTEN mRNA was analyzed in goldstriped amberjack Seriola aureovittata by quantitative PCR(qPCR) to explore the molecular mechanism of PTEN in the growth and development of goldstriped amberjack. The results showed that the PTEN cDNA sequence was 1575 bp in length and the ORF was 1287 bp, encoding 428 amino acids. PTEN has the common feature of vertebrate PTEN and contains PTP(PTPc motif) and PTEN-C2 domain. Sequence alignment analysis revealed that both goldstriped amberjack and greater amberjack Seriola dumerili had a high homology(95%). The phylogenetic tree indicated that goldstriped amberjack PTEN was clustered into one branch with the PTEN Perciformes and Pleuronectiformes.By qPCR analysis, the maximal relative expression of PTEN was observed in the heart of goldstriped amberjack(P<0.05), followed by in the spleen, head kidney, kidney, stomach, intestine, gill and pituitary, indicating that the PTEN is involved in the regulation of a variety of physiological processes in various tissues due to high expression of PTEN mRNA. It was found that PTEN was continuously expressed in early embryonic development(embryonic stage, larvae and juvenile), with significantly higher expression level from early blastula stage(38 hours post fertilization, hpf)(P<0.05) with the maximal expression level in freshly hatched larvae(P<0.05). The expression level of PTEN mRNA was gradually decreased during the postlarvae and juvenile(P<0.05). The expression level of PTEN was found to be gradually decreased, significantly and constantly low at 35 days old. The findings indicate that PTEN plays an important regulatory role in the development of embryos and juveniles, which provides a theoretical basis with research of the regulation mechanism and culture during the early growth and development stages in goldstriped amberjack.
引文
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[15] Lee J O,Yang Haijuan,Georgescu M M,et al.Crystal structure of the PTEN tumor suppressor:implications for its phosphoinositide phosphatase activity and membrane association[J].Cell,1999,99(3):323-334.
[16] Planchon S M,Waite K A,Eng C.The nuclear affairs of PTEN[J].Journal of Cell Science,2008,121:249-253.
[17] Wijesekara N,Konrad D,Eweida M,et al.Muscle-specific Ptendeletion protects against insulin resistance and diabetes[J].Molecular and Cellular Biology,2005,25(3):1135-1145.
[18] Hu Zhaoyong,Wang Huiling,Lee I H,et al.PTEN inhibition improves muscle regeneration in mice fed a high-fat diet[J].Diabetes,2010,59(6):1312-1320.
[19] Feng Yue,Bi Pengpeng,Wang Chao,et al.Pten is necessary for the quiescence and maintenance of adult muscle stem cells[J].Nature Communications,2017,8:14328.
[20] Halet G,Viard P,Carroll J.Constitutive PtdIns(3,4,5)P3 synthesis promotes the development and survival of early mammalian embryos[J].Development,2008,135(3):425-429.
[21] 王喜宏,和协超,韩树标,等.PTEN在小鼠卵细胞和早期胚胎发育中的功能研究[J].动物学研究,2011,32(6):647-650.
[22] Jagarlamudi K,Liu Lian,Adhikari D,et al.Oocyte-specific deletion of Pten in mice reveals a stage-specific function of PTEN/PI3K signaling in oocytes in controlling follicular activation[J].PLoS One,2009,4(7):e6186.
[23] Yang S G,Hur S W,Ji S C,et al.Morphological development of embryo,larvae and juvenile in yellowtail kingfish,Seriola lalandi[J].Development & Reproduction,2016,20(2):131-140.
[24] Martínez-Montaňo E,González-álvarez K,Lazo J P,et al.Morphological development and allometric growth of yellowtail kingfish Seriola lalandi V.larvae under culture conditions[J].Aquaculture Research,2016,47(4):1277-1287.
[2] Furnari F B,Huang H J S,Cavenee W K.The phosphoinositol phosphatase activity of PTEN mediates a serum-sensitive G1 growth arrest in glioma cells[J].Cancer Research,1998,58(22):5002-5008.
[3] Xu Zheng,Stokoe D,Kane L P,et al.The inducible expression of the tumor suppressor gene PTEN promotes apoptosis and decreases cell size by inhibiting the PI3K/Akt pathway in Jurkat T cells[J].Cell Growth & Differentiation,2002,13(7):285-296.
[4] Rhei E,Kang Lan,Bogomolniy F,et al.Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in primary breast carcinomas[J].Cancer Research,1997,57(17):3657-3659.
[5] Dupont J,Renou J P,Shani M,et al.PTEN overexpression suppresses proliferation and differentiation and enhances apoptosis of the mouse mammary epithelium[J].The Journal of Clinical Investigation,2002,110(6):815-825.
[6] Podsypanina K,Ellenson L H,Nemes A,et al.Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(4):1563-1568.
[7] Reddy P,Liu Lian,Adhikari D,et al.Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool[J].Science,2008,319(5863):611-613.
[8] Stumpf M,Choorapoikayil S,Den Hertog J.Pten function in zebrafish:anything but a fish story[J].Methods,2015,77-78:191-196.
[9] Croushore J A,Blasiole B,Riddle R C,et al.Ptena and ptenb genes play distinct roles in zebrafish embryogenesis[J].Developmental Dynamics,2010,234(4):911-921.
[10] 柳学周,徐永江,李荣,等.黄条鰤(Seriola aureovittata)肌肉营养组成分析与评价[J].渔业科学进展,2017,38(1):128-135.
[11] 史宝,刘永山,柳学周,等.黄条鰤(Seriola aureovittata)染色体核型分析[J].渔业科学进展,2017,38(1):136-141.
[12] 钟建兴,蔡良候,郑惠东,等.黄条鰤胚胎发育观察[J].福建水产,2010(2):22-25.
[13] Tom M,Chen N,Segev M,et al.Quantifying fish metallothionein transcript by real time PCR for its utilization as an environmental biomarker[J].Marine Pollution Bulletin,2004,48(7-8):705-710.
[14] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408.
[15] Lee J O,Yang Haijuan,Georgescu M M,et al.Crystal structure of the PTEN tumor suppressor:implications for its phosphoinositide phosphatase activity and membrane association[J].Cell,1999,99(3):323-334.
[16] Planchon S M,Waite K A,Eng C.The nuclear affairs of PTEN[J].Journal of Cell Science,2008,121:249-253.
[17] Wijesekara N,Konrad D,Eweida M,et al.Muscle-specific Ptendeletion protects against insulin resistance and diabetes[J].Molecular and Cellular Biology,2005,25(3):1135-1145.
[18] Hu Zhaoyong,Wang Huiling,Lee I H,et al.PTEN inhibition improves muscle regeneration in mice fed a high-fat diet[J].Diabetes,2010,59(6):1312-1320.
[19] Feng Yue,Bi Pengpeng,Wang Chao,et al.Pten is necessary for the quiescence and maintenance of adult muscle stem cells[J].Nature Communications,2017,8:14328.
[20] Halet G,Viard P,Carroll J.Constitutive PtdIns(3,4,5)P3 synthesis promotes the development and survival of early mammalian embryos[J].Development,2008,135(3):425-429.
[21] 王喜宏,和协超,韩树标,等.PTEN在小鼠卵细胞和早期胚胎发育中的功能研究[J].动物学研究,2011,32(6):647-650.
[22] Jagarlamudi K,Liu Lian,Adhikari D,et al.Oocyte-specific deletion of Pten in mice reveals a stage-specific function of PTEN/PI3K signaling in oocytes in controlling follicular activation[J].PLoS One,2009,4(7):e6186.
[23] Yang S G,Hur S W,Ji S C,et al.Morphological development of embryo,larvae and juvenile in yellowtail kingfish,Seriola lalandi[J].Development & Reproduction,2016,20(2):131-140.
[24] Martínez-Montaňo E,González-álvarez K,Lazo J P,et al.Morphological development and allometric growth of yellowtail kingfish Seriola lalandi V.larvae under culture conditions[J].Aquaculture Research,2016,47(4):1277-1287.