香港牡蛎Sox8基因克隆分析与表达模式研究
|
摘要
应用生物信息学方法和QRT-PCR技术,系统分析和预测香港牡蛎Sox8基因的遗传进化、蛋白质的理化性质和结构特征及组织表达差异分析。结果表明:香港牡蛎Sox8基因编码区全长1374bp,共编码氨基酸457个。多重序列比较分析显示香港牡蛎Sox8核苷酸序列相似性与太平洋牡蛎最高,为96%,系统进化树分析显示在不同物种以及进化的过程中Sox8基因具有较高的保守性。Sox8蛋白质信息学分析结果表明该蛋白呈弱碱性,N端无信号肽存在,位于胞核中,含有HMG-box和Sox_N等结构。定量分析结果显示:Sox8在香港牡蛎8种组织中均有表达,其中表达量最高的是血淋巴,其次是性腺和唇瓣,鳃中相对表达量最低。研究结果为今后阐明其在香港牡蛎性腺发育进程中的功能奠定了基础。
Bio-informatics techniques and QRT-PCR was used to analysis the gene sequence character and protein structure,the expression pattern of crassostrea hongkongensis Sox8 in different tissues.The results showed that a 1608 bp crassostrea hongkongensis Sox8 gene fragment including a 1374 bp whole length CDS(coded 457 amino acids) was cloned and sequenced.The sequence multi-aligned results showed that crassostrea hongkongensis Sox8 gene shared 96%of similar nucleotide sequence with that of crassostrea gigas.Sox8 protein sequence analysis showed that it was weakly alkaline,hadn't signal peptide,positioned in the nuclear,and with the presence of HMG-box and Sox_N structure.In addition,the expression pattern analysis results showed that crassostrea hongkongensis Sox8 expressed in mantle,gill,adductor muscle,gonad,visceral mass,labial palp,heart and hemocytes etc.8 tested samples,with the most abundant expression in hemocytes,followed by gonad and labial palp,the minimal expression in gill was observed.The cloning and analysis of Sox8 gene provided an important foundation for further study reproduction regulation mechanism of Sox8 gene in Crassostrea hongkongensis in the future.
Bio-informatics techniques and QRT-PCR was used to analysis the gene sequence character and protein structure,the expression pattern of crassostrea hongkongensis Sox8 in different tissues.The results showed that a 1608 bp crassostrea hongkongensis Sox8 gene fragment including a 1374 bp whole length CDS(coded 457 amino acids) was cloned and sequenced.The sequence multi-aligned results showed that crassostrea hongkongensis Sox8 gene shared 96%of similar nucleotide sequence with that of crassostrea gigas.Sox8 protein sequence analysis showed that it was weakly alkaline,hadn't signal peptide,positioned in the nuclear,and with the presence of HMG-box and Sox_N structure.In addition,the expression pattern analysis results showed that crassostrea hongkongensis Sox8 expressed in mantle,gill,adductor muscle,gonad,visceral mass,labial palp,heart and hemocytes etc.8 tested samples,with the most abundant expression in hemocytes,followed by gonad and labial palp,the minimal expression in gill was observed.The cloning and analysis of Sox8 gene provided an important foundation for further study reproduction regulation mechanism of Sox8 gene in Crassostrea hongkongensis in the future.
引文
[1] 李永婧,吴利敏,李学军.硬骨鱼类性别决定与分化相关基因研究进展[J].河南师范大学学报(自然版),2017(4):72-78.
[2] 路畅,苏利娜,朱邦科.鱼类性别决定及分化相关基因研究进展[J].湖北农业科学,2014(13):2981-2986.
[3] BOWLES J,SCHEPERS G,KOOPMAN P.Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators[J].Developmental Biology,2000,227(2):239-255.
[4] 高进.半滑舌鳎(Cynoglossus semilaevis)性腺发育中性别相关基因挖掘及Sox基因家族生物信息学分析[D].南京:南京农业大学,2016.
[5] 高进,马佳璐,刘洋,等.半滑舌鳎(Cynoglossus semilaevis)Sox基因家族生物信息学分析[J].渔业科学进展,2016(2):41-48.
[6] 杨超.尼罗罗非鱼Sox家族生物信息学分析及Sox9α/b在性腺发育中的可能作用[D].重庆:西南大学,2013.
[7] CUI J,SHEN X,ZHAO H,et al.Genome-wide analysis of Sox genes in Medaka (Oryzias latipes) and their expression pattern in embryonic development[J].Cytogenetic& Genome Research,2011,134(4):283-294.
[8] SHE Z Y,YANG W X.SOX family transcription factors involved in diverse cellular events during development[J].European Journal of Cell Biology,2015,94(12):547-563.
[9] CHABOISSIER M C,KOBAYASHI A,VIDAL V I,etal.Functional analysis of Sox8 and Sox9 during sex determination in the mouse[J].Development,2004,131(9):1891-1901.
[10] KIEFER J C.Back to basics:Sox genes[J].Developmental Dynamics,2007,236(8):2356-2366.
[11] TONG Y,ZHANG Y,HUANG J,et al.Transcriptomics Analysis of Crassostrea hongkongensis for the Discovery of Reproduction-Related Genes[J].Plos One,2015,10(8):e 0134280.
[12] SANTERRE C,SOURDAINE P,ADELINE B,et al.Cg-SoxE and Cg-β-catenin,two new potential actors of the sex-determining pathway in a hermaphrodite lophotrochozoan,the Pacific oyster Crassostrea gigas[J].Comparative Biochemistry & Physiology Part A Molecular & Integrative Physiology,2014,167(167):68-76.
[13] 秦宏敏,殷光义,梁淑芳,等.高泳动家族性别决定基因9(Sox9)启动子转染软骨肉瘤细胞的实验研究[J].中国矫形外科杂志,2015(15):1411-1414.
[14] 曹慧慧,王昌留.Sox基因家族特点及其功能[J].鲁东大学学报(自然科学版),2011(1):58-63.
[15] 刘昌海,李琪,孔令锋.太平洋牡蛎内切葡聚糖酶基因的克隆、序列特性和表达分析[J].中国海洋大学学报(自然科学版),2012,42(S1):87-93.
[16] 秦国强.SOX家族基因在前列腺癌进展及预后上的重要作用[D].广州:南方医科大学,2012.
[17] 周祖阳,李琪,于红,等.长牡蛎Fem-1基因cDNA克隆和表达分析[J].中国海洋大学学报(自然科学版),2018,48(6):45-54.
[18] LETUNIC I,BORK P.20 years of the SMART protein domain annotation resource[J].Nucleic Acids Res,2018,46(D1):D493-D496.
[19] NIELSEN H.Predicting Secretory Proteins with SignalP[J].Methods Mol Biol,2017(1611):59-73.
[20] YANG J,YAN R,ROY A,et al.The I-TASSER Suite:protein structure and function prediction[J].Nat Methods,2015,12(1):7-8.
[21] 岳素文.肝癌发生中SOX家族基因表达异常研究[D].郑州:郑州大学,2011.
[22] 常重杰,杜启艳,周荣家,等.具有内含子的大鳞副泥鳅Sox8基因(英文)[J].Developmental & Reproductive Biology,2000(2):15-22.
[23] 任莉莉,龚炜,周荣家,等.Sox8基因HMG盒区内含子剪接位点分析[J].遗传,2001(4):298-300.
[24] PORTNOI M F,DUMARGNE M C,ROJO S,et al.Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies[J].Human Molecular Genetics,2018,27(7):1228-1240.
[25] BARRIONUEVO F J,HURTADO A,KIM G J,et al.Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration[J].Elife,2016:e15635.
[26] 于非非,王梅芳,桂建芳,等.马氏珠母贝Sox11基因的克隆及时序表达模式分析[J].水生生物学报,2016(1):71-75.
[27] 许开航,王梅芳,余祥勇,等.企鹅珍珠贝Sox9基因的克隆及表达分析[J].广东海洋大学学报,2018(2):15-22.
[28] 张娜.长牡蛎的性别决定机制及三倍体性腺发育的分子基础研究[D].北京:中国科学院大学,2014.
[29] 谢超.SOX8在非小细胞型肺癌中的表达及miRNA-124治疗作用的研究[D].济南:山东大学,2015.
[2] 路畅,苏利娜,朱邦科.鱼类性别决定及分化相关基因研究进展[J].湖北农业科学,2014(13):2981-2986.
[3] BOWLES J,SCHEPERS G,KOOPMAN P.Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators[J].Developmental Biology,2000,227(2):239-255.
[4] 高进.半滑舌鳎(Cynoglossus semilaevis)性腺发育中性别相关基因挖掘及Sox基因家族生物信息学分析[D].南京:南京农业大学,2016.
[5] 高进,马佳璐,刘洋,等.半滑舌鳎(Cynoglossus semilaevis)Sox基因家族生物信息学分析[J].渔业科学进展,2016(2):41-48.
[6] 杨超.尼罗罗非鱼Sox家族生物信息学分析及Sox9α/b在性腺发育中的可能作用[D].重庆:西南大学,2013.
[7] CUI J,SHEN X,ZHAO H,et al.Genome-wide analysis of Sox genes in Medaka (Oryzias latipes) and their expression pattern in embryonic development[J].Cytogenetic& Genome Research,2011,134(4):283-294.
[8] SHE Z Y,YANG W X.SOX family transcription factors involved in diverse cellular events during development[J].European Journal of Cell Biology,2015,94(12):547-563.
[9] CHABOISSIER M C,KOBAYASHI A,VIDAL V I,etal.Functional analysis of Sox8 and Sox9 during sex determination in the mouse[J].Development,2004,131(9):1891-1901.
[10] KIEFER J C.Back to basics:Sox genes[J].Developmental Dynamics,2007,236(8):2356-2366.
[11] TONG Y,ZHANG Y,HUANG J,et al.Transcriptomics Analysis of Crassostrea hongkongensis for the Discovery of Reproduction-Related Genes[J].Plos One,2015,10(8):e 0134280.
[12] SANTERRE C,SOURDAINE P,ADELINE B,et al.Cg-SoxE and Cg-β-catenin,two new potential actors of the sex-determining pathway in a hermaphrodite lophotrochozoan,the Pacific oyster Crassostrea gigas[J].Comparative Biochemistry & Physiology Part A Molecular & Integrative Physiology,2014,167(167):68-76.
[13] 秦宏敏,殷光义,梁淑芳,等.高泳动家族性别决定基因9(Sox9)启动子转染软骨肉瘤细胞的实验研究[J].中国矫形外科杂志,2015(15):1411-1414.
[14] 曹慧慧,王昌留.Sox基因家族特点及其功能[J].鲁东大学学报(自然科学版),2011(1):58-63.
[15] 刘昌海,李琪,孔令锋.太平洋牡蛎内切葡聚糖酶基因的克隆、序列特性和表达分析[J].中国海洋大学学报(自然科学版),2012,42(S1):87-93.
[16] 秦国强.SOX家族基因在前列腺癌进展及预后上的重要作用[D].广州:南方医科大学,2012.
[17] 周祖阳,李琪,于红,等.长牡蛎Fem-1基因cDNA克隆和表达分析[J].中国海洋大学学报(自然科学版),2018,48(6):45-54.
[18] LETUNIC I,BORK P.20 years of the SMART protein domain annotation resource[J].Nucleic Acids Res,2018,46(D1):D493-D496.
[19] NIELSEN H.Predicting Secretory Proteins with SignalP[J].Methods Mol Biol,2017(1611):59-73.
[20] YANG J,YAN R,ROY A,et al.The I-TASSER Suite:protein structure and function prediction[J].Nat Methods,2015,12(1):7-8.
[21] 岳素文.肝癌发生中SOX家族基因表达异常研究[D].郑州:郑州大学,2011.
[22] 常重杰,杜启艳,周荣家,等.具有内含子的大鳞副泥鳅Sox8基因(英文)[J].Developmental & Reproductive Biology,2000(2):15-22.
[23] 任莉莉,龚炜,周荣家,等.Sox8基因HMG盒区内含子剪接位点分析[J].遗传,2001(4):298-300.
[24] PORTNOI M F,DUMARGNE M C,ROJO S,et al.Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies[J].Human Molecular Genetics,2018,27(7):1228-1240.
[25] BARRIONUEVO F J,HURTADO A,KIM G J,et al.Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration[J].Elife,2016:e15635.
[26] 于非非,王梅芳,桂建芳,等.马氏珠母贝Sox11基因的克隆及时序表达模式分析[J].水生生物学报,2016(1):71-75.
[27] 许开航,王梅芳,余祥勇,等.企鹅珍珠贝Sox9基因的克隆及表达分析[J].广东海洋大学学报,2018(2):15-22.
[28] 张娜.长牡蛎的性别决定机制及三倍体性腺发育的分子基础研究[D].北京:中国科学院大学,2014.
[29] 谢超.SOX8在非小细胞型肺癌中的表达及miRNA-124治疗作用的研究[D].济南:山东大学,2015.