泥鳅cyp19a1基因克隆及其在倍性间的表达差异
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国家重点研发计划“蓝色粮仓科技创新”重点专项(2018YFD0900205)


Molecular cloning and differential expression of cyp19a1 in tetraploid and diploid loach,Misgurnus anguillicaudatus
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    摘要:

    克隆并分析泥鳅cyp19a1a的全长cDNA和5′侧翼序列,用qRT-PCR技术比较cyp19a1a在二倍体、四倍体泥鳅组织间和倍性间的表达差异。结果发现,泥鳅cyp19a1a的cDNA全长1 905 bp,包括29 bp的5′TR、301 bp的3′UTR和1 575 bp的ORF序列,其氨基酸序列中存在I-螺旋区、芳香化酶特异保守区以及血红素结合区等重要功能域。用hiTAIL-PCR克隆获得2 040 bp的5′侧翼序列,在该序列上预测到典型元件TATA-box,以及C/EBPβ、SRY、ER、CREB、GR等转录因子结合位点。12月龄四倍体泥鳅的体长、体质量均显著高于二倍体,但性腺发育明显滞后于二倍体。cyp19a1a和cyp19a1b分别在二倍体、四倍体泥鳅的性腺和脑中的表达量最高。倍性间比较结果显示,cyp19a1a在四倍体各组织中的表达均高于二倍体(除精巢外);cyp19a1b在四倍体雌鳅脑中的表达量显著高于二倍体,但在四倍体雄鳅脑中的表达量显著低于二倍体。综合分析上述结果,四倍体中相对较高的cyp19a1a表达可能与其性腺所处发育时期有关,较晚的性成熟有利于泥鳅将更多能量用于个体生长。由于cyp19a1及其催化产生的雌激素还可以通过GH-IGF通路调节鱼类的生长,推测cyp19a1在泥鳅倍性间的差异表达可能与二倍体、四倍体间的生长生殖差异密切相关。

    Abstract:

    Tetraploid loach (Misgurnus anguillicaudatus) exhibits superior growth performance but delayed reproductive development when compared with diploid individuals. To understand the potential molecular mechanism of this physiological difference between-ploidy,the full-length cDNA and 5′-flanking sequence of cyp19a1a in loach were cloned and analyzed,and the expression of cyp19a1a between-ploidy were compared using qRT-PCR method. The results showed that,the full-length cDNA of cyp19a1a was 1 905 bp,which contained a 29 bp 5′UTR,301 bp 3′UTR and 1 575 bp ORF. Some important functional domains were predicted in the cyp19a1a protein,such as I-helix region,aromatase specific conservative region and heme-binding region. Using hiTAIL-PCR method,a 2 040 bp 5′-flanking sequence was cloned,and the typical function element TATA-box,as well as some important transcription factor binding sites such as SP1,AP-1,C/EBPβ,SRY,ER,CREB,GR were predicted. At the age of 12 month,the body length and weight of tetraploids were significantly higher than those of diploids loach (P<0.05). However,the gonadal development of tetraploids was delayed obviously. In both diploids and tetraploids,the cyp19a1a expression was the highest in the gonad,while cyp19a1b was the highest in the brain. The loach cyp19a1a exhibited higher expression in tetraploids than in diploids in all tissues except for the testis. The cyp19a1b expression in the brain was significantly higher in tetraploid female than in diploid female,while an opposite result was observed in male (P<0.05). Combining all results obtained in the present study,it was suggested that the higher expression of cyp19a1a in tetraploids should be due to the special developmental stage of the gonads,and the delayed sexual maturity in tetraploids may partially contribute to its superior growth performance. In addition,as cyp19a1a and estrogen also regulate fish growth through the GH-IGF pathway,it is speculated that the differential expression of cyp19a1a between-ploidy might be related to the different growth performance and reproductive development of diploid and tetraploid loach.

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曹文怡,易少奎,杨楠,苏君晓,罗双双,高泽霞,周小云.泥鳅cyp19a1基因克隆及其在倍性间的表达差异[J].华中农业大学学报,2021,40(4):166-176

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  • 收稿日期:2021-02-25
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  • 在线发布日期: 2021-07-22
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