中华鳖NR5a2基因的克隆与表达分析
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摘要
为了解NR5a2在中华鳖(Pelodiscus sinensis)性腺分化过程中的作用,通过RACE技术克隆获得中华鳖NR5a2 cDNA。结果显示:该基因序列全长为2 674 bp,开放阅读框为1 608 bp,编码535个氨基酸,5′非编码区长度为40 bp,3′非编码区长度为1 026 bp。生物信息学分析表明该基因编码的蛋白质分子质量为60.5 ku,等电点为8.57,具有保守的ZnF-C4结构域和HOLI结构域。氨基酸多重序列比对分析表明中华鳖NR5a2基因与西部锦龟(Chrysemys picta bellii)的相似性最高,其次是原鸡(Gallus gallus)。系统进化分析表明中华鳖NR5a2与西部锦龟的亲缘关系最近。实时荧光定量PCR结果表明NR5a2在中华鳖不同组织中均有表达,且卵巢组织中的表达量显著高于精巢组织。31.5℃孵化条件下,不同发育时期的中华鳖胚胎组织中发现NR5a2基因在17期时表达量达到最高,随后显著下降。结果表明NR5a2可能参与中华鳖的性别分化。
The complete nucleotide sequence of NR5a2 cDNA in Pelodiscus sinensis was obtained by 3′RACE and 5′RACE in present study to understand the effect of specific gene on gonadal differentiation.The full-length cDNA of NR5a2 contained 2 674 bp.The open reading frame(ORF)was 1 608 bp,encoding a putative protein of 535 amino acids.The length of 5′-UTR and 3′-URT was 40 bp and 1 026 bp,respectively.Bioinformatics analysis showed that the molecular weight of the protein encoded by the NR5a2 was 60.5 ku,and the isoelectric point was 8.57.The ORF contained one conservative ZnF-C4 domain and one conservative HOLI domain.Multiple sequence alignment analysis revealed that the NR5a2 coding region showed mostly similarity in amino acids sequence with Chrysemys picta bellii,followed by Gallus gallus.Phylogenetic tree analysis showed that P.sinensis was more affinity with C.picta bellii.The qRT-PCR analysis showed that NR5a2 was existed in all the examined tissues,and the expression level of ovarian tissue was significantly higher than that of testis.The expression of NR5a2 in the 17 th stage of embryo development was the highest and then significantly decreased.Therefore,the NR5a2 gene might be involved in gender differentiation of P.sinensis.
The complete nucleotide sequence of NR5a2 cDNA in Pelodiscus sinensis was obtained by 3′RACE and 5′RACE in present study to understand the effect of specific gene on gonadal differentiation.The full-length cDNA of NR5a2 contained 2 674 bp.The open reading frame(ORF)was 1 608 bp,encoding a putative protein of 535 amino acids.The length of 5′-UTR and 3′-URT was 40 bp and 1 026 bp,respectively.Bioinformatics analysis showed that the molecular weight of the protein encoded by the NR5a2 was 60.5 ku,and the isoelectric point was 8.57.The ORF contained one conservative ZnF-C4 domain and one conservative HOLI domain.Multiple sequence alignment analysis revealed that the NR5a2 coding region showed mostly similarity in amino acids sequence with Chrysemys picta bellii,followed by Gallus gallus.Phylogenetic tree analysis showed that P.sinensis was more affinity with C.picta bellii.The qRT-PCR analysis showed that NR5a2 was existed in all the examined tissues,and the expression level of ovarian tissue was significantly higher than that of testis.The expression of NR5a2 in the 17 th stage of embryo development was the highest and then significantly decreased.Therefore,the NR5a2 gene might be involved in gender differentiation of P.sinensis.
引文
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[15]顾月琴,张伟,王利红.小鼠LRH-1基因CDS区序列克隆及分析[J].安徽农业大学学报,2011,38(3):372-375.
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[17] Duggavathi R,Volle D H,Mataki C,et al.Liver receptor homolog 1 is essential for ovulation [J].Gene Dev,2008,22(14):1871-1876.
[18] Kanda H,Okubo T,Omori N,et al.Transcriptional regulation of the rainbow trout Cyp19a gene by Ftz-f1 homologue [J].J Steroid Biochem Mol Biol,2006,99(2-3):85-92.
[19] Wang L,You F,Weng S,et al.Molecular cloning and sexually dimorphic expression patterns of Nr0b1 and NR5a2 in olive flounder,Paralichthys olivaceus [J].Dev Genes Evol,2015,225(2):95-104.
[20] Yan H,Shen X,Cui X,et al.Identification of genes involved in gonadal sex differentiation and the dimorphic expression pattern in Takifugu rubripes,gonad at the early stage of sex differentiation [J].Fish Physiol Biochem,2018,44(5):1275-1290.
[2]范兆飞.牙鲆cyp19a及其转录因子表观修饰和调控研究[D].北京:中国科学院大学(中国科学院海洋研究所),2017.
[3] Lu H,Zhang S,Liu Q,et al.Cytoplasmic localization of Lrh-1 down-regulates ovarian follicular Cyp19a1a expression in a teleost,the orange-spotted grouper epinephelus coioides [J].Biol Reprod,2014,91(2):29.
[4] Shi B,Lu H,Zhang L,et al.A homologue of NR5a1 activates Cyp19a1a transcription additively with NR5a2 in ovarian follicular cells of the orange-spotted grouper [J].Mol Cell Endocrinol,2017,460:85-93.
[5]赵淑梅.鳖病的综合防治[J].淡水渔业,2000,30(1):37-39.
[6]罗晓松,刘志刚.中华鳖出血性肠道坏死症及中草药防治实验[J].淡水渔业,2000,30(7):46-47.
[7]覃川杰,汪成竹,陈晓辉,等.茯苓多糖对中华鳖非特异性免疫功能的免疫调节作用[J].淡水渔业,2006,36(6):40-43.
[8]王莉.中华鳖DMRT1基因在雄性性腺发育中的影响研究[D].上海:上海海洋大学,2014.
[9] Tokita M,Kuratani S.Normal embryonic stages of the chinese softshelled turtle Pelodiscus sinensis(Trionychidae )[J].Zool Sci,2001,18(5):705-715.
[10] Chutian G E,Sun W,Sang Y P,et al.Molecular cloning and expression pattern of Dmrt1 and its expression change in sex reversal in Pelodiscus sinensis [J].Scientia Sinica Vitae,2014,44(11):1173-1182.
[11]王莉,孙伟,储经乾,等.中华鳖SOX9基因在胚胎期性腺中的表达模式及在性逆转中的表达变化[J].水产学报,2014,38(9):1286-1293.
[12]包海声,蔡晗,韩伟,等.Cyp19a1基因在中华鳖早期卵巢分化中的功能研究[J].中国科学:生命科学,2017,47(6):640-649.
[13]姚勇,潘增祥,张久峰,等.二花脸猪NR5a2基因克隆与卵巢组织转录水平分析[J].南京农业大学学报,2013,36(3):133-138.
[14]唐永凯,俞菊华,徐跑,等.黄颡鱼LRH-1基因的分离和表达[J].中国水产科学,2009,16(5):649-659.
[15]顾月琴,张伟,王利红.小鼠LRH-1基因CDS区序列克隆及分析[J].安徽农业大学学报,2011,38(3):372-375.
[16] Falender A E,Lanz R,Malenfant D,et al.Differential expression of steroidogenic factor-1 and Ftf/Lrh-1 in the rodent ovary [J].Endocrinology,2003,144(8):3598-3610.
[17] Duggavathi R,Volle D H,Mataki C,et al.Liver receptor homolog 1 is essential for ovulation [J].Gene Dev,2008,22(14):1871-1876.
[18] Kanda H,Okubo T,Omori N,et al.Transcriptional regulation of the rainbow trout Cyp19a gene by Ftz-f1 homologue [J].J Steroid Biochem Mol Biol,2006,99(2-3):85-92.
[19] Wang L,You F,Weng S,et al.Molecular cloning and sexually dimorphic expression patterns of Nr0b1 and NR5a2 in olive flounder,Paralichthys olivaceus [J].Dev Genes Evol,2015,225(2):95-104.
[20] Yan H,Shen X,Cui X,et al.Identification of genes involved in gonadal sex differentiation and the dimorphic expression pattern in Takifugu rubripes,gonad at the early stage of sex differentiation [J].Fish Physiol Biochem,2018,44(5):1275-1290.