Fish of the superfamily Cobitoidea sensu stricto （namely Ioaches） exhibit extremely high diversity of color patterns, but so far little is known about their evolutionary mechanism. Melanocortin 1 receptor gene （MCI...Fish of the superfamily Cobitoidea sensu stricto （namely Ioaches） exhibit extremely high diversity of color patterns, but so far little is known about their evolutionary mechanism. Melanocortin 1 receptor gene （MCIR） plays an important role during the synthesis of melanin and formation of animal body color patterns. In this study, we amplified and sequenced the partial MCIR gene for 44 loach individuals representing 31 species of four families. Phylogenetic analyses yielded a topology congruent with previous studies using multiple nuclear loci, showing that each of the four families was monophyletic with sister relationships of Botiidae＋ （Cobitidae＋（Balitoridae＋Nemacheilidae））. Gene evolutionary analyses indicated that MCIR in Ioaches was under purifying selection pressure, with various sites having different dNIds values. Both Botiidae and Cobitidae had lower dN/ds values than those of background lineages, suggesting their evolution might be strongly affected by purifying selection pressure. For Balitoddae and Nemacheilidae, both had larger dNIds values than those of background lineages, suggesting they had a faster evolutionary rate under more relaxed selection pressure. Consequently, we inferred that the relatively stable color patterns in Botiidae and Cobitidae might result from the strong purifying selection pressure on the MC1R gene, whereas the complicated and diverse color patterns in Balitoridae and Nemacheilidae might be associated with the relaxed selection pressure. Given the easy experimental procedure for the partial MCTR gene and its excellent performance in reconstructing phylogeny, we suggest this gene could be used as a good molecular marker for the phylogenetic study of fish species.展开更多
文摘以小干扰RNA(siRNA)技术抑制恶性黑素瘤细胞株A375的黑素皮质素受体1(melanocortin-1-receptor,MC1R)、小眼球相关转录因子(microphthalmia-associated transcription factor,MITF)基因表达,从而抑制细胞黑色素的生成。通过RT-PCR检测MC1R、MITF、酪氨酸酶(tyrosinase,TYR)、酪氨酸酶相关蛋白l(tyrosinase related protein-1,TRP-1)基因mRNA水平的变化,通过测定黑色素含量的变化检测蛋白水平的改变。并用台盼蓝活细胞计数法检测其对细胞增殖的影响。经RT-PCR检测,特异性siRNA作用的靶基因显著下调,黑色素含量明显降低。细胞增殖的变化说明,脂质体包裹的siRNA对A375细胞的毒性远小于熊果苷等化学合成药物。结果显示,利用siRNA对A375细胞中靶基因的调控,有效的抑制了黑色素的生成,siRNA技术与传统的调控黑色素的方法(化学合成物——褪色剂)比较,具有用量小、抑制效率高、细胞毒性低等优点。同时,为进一步研究黑色素形成通路中各基因之间的的关系提供了新思路。
基金supported by the National Natural Science Foundation of China(NSFC 31272306,31400359,31401968)
文摘Fish of the superfamily Cobitoidea sensu stricto （namely Ioaches） exhibit extremely high diversity of color patterns, but so far little is known about their evolutionary mechanism. Melanocortin 1 receptor gene （MCIR） plays an important role during the synthesis of melanin and formation of animal body color patterns. In this study, we amplified and sequenced the partial MCIR gene for 44 loach individuals representing 31 species of four families. Phylogenetic analyses yielded a topology congruent with previous studies using multiple nuclear loci, showing that each of the four families was monophyletic with sister relationships of Botiidae＋ （Cobitidae＋（Balitoridae＋Nemacheilidae））. Gene evolutionary analyses indicated that MCIR in Ioaches was under purifying selection pressure, with various sites having different dNIds values. Both Botiidae and Cobitidae had lower dN/ds values than those of background lineages, suggesting their evolution might be strongly affected by purifying selection pressure. For Balitoddae and Nemacheilidae, both had larger dNIds values than those of background lineages, suggesting they had a faster evolutionary rate under more relaxed selection pressure. Consequently, we inferred that the relatively stable color patterns in Botiidae and Cobitidae might result from the strong purifying selection pressure on the MC1R gene, whereas the complicated and diverse color patterns in Balitoridae and Nemacheilidae might be associated with the relaxed selection pressure. Given the easy experimental procedure for the partial MCTR gene and its excellent performance in reconstructing phylogeny, we suggest this gene could be used as a good molecular marker for the phylogenetic study of fish species.
