莆田黑鸭MITF基因表达特性及其与黑色素沉积的关联分析  

Expression of MITF Gene in Putian Black Duck(Anas anas domesticus)and Its Association with Melanin Deposition

作　　者：辛清武[1] 李丽[1] 缪中纬[1] 章琳俐[1] 刘艺展 朱志明[1] 郑嫩珠[1] XIN Qing-Wu;LI Li;MIAO Zhong-Wei;ZHANG Lin-Li;LIU Yi-Zhan;ZHU Zhi-Ming;ZHENG Nen-Zhu(Institute of Animal Science and Veterinary Medicine,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China;College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建省农科院畜牧兽医研究所,福州350013 [2]福建农林大学动物科技学院,福州350002

出　　处：《农业生物技术学报》2018年第11期1928-1937,共10页Journal of Agricultural Biotechnology

基　　金：福建省属公益类科研院所专项(No.2018R1023-2);福建省农科院创新团队项目(No.STTT2017-2-1);福建省种业创新与产业化工程项目(No.fjzycxny2017011-01)

摘　　要：莆田黑鸭(Anas anas domesticus)是我国唯一的黑羽蛋鸭地方品种,为中国珍贵稀有的水禽资源。小眼畸形相关转录因子(microphthalmia-associtated transcription factor, MITF)作为黑色素细胞最重要的转录因子,可通过α-MSH (melanocyte-stimulating hormone)诱导,与cAMP应答原件结合蛋白(cAMP responsive element binding protein, CREB)结合作用于cAMP,激活MITF基因在黑色素细胞中特异表达,对于黑色素的合成具有关键作用。为探讨莆田黑鸭各组织MITF基因的表达特性,及其与真黑色素在莆田黑鸭各组织中沉积规律的相关性,本研究利用巢式PCR方法,首次从莆田黑鸭皮肤组织克隆MITF基因(GenBank No. MG516571),并进行了序列分析。进而利用实时荧光定量PCR方法检测莆田黑鸭各组织的MITF基因表达,利用紫外分光光度法测定莆田黑鸭各组织真黑色素的沉积量,应用线性相关分析方法分析莆田黑鸭各组织MITF基因表达与真黑色素沉积的相关性。结果表明,莆田黑鸭MITF基因长度为1 341bp,包含完整的CDS序列(12~1334 bp);编码440个氨基酸,与绿头鸭(Anas platyrhynchos)的氨基酸同源性为100%,与原鸡(Gallus gallus)、鹌鹑(Coturnix japonica)等鸟类的氨基酸同源性在95%以上。遗传进化分析发现,莆田黑鸭MITF与绿头鸭的亲缘关系最近,与鸿雁(Anser cygnoides)、白绒乌鸡(Silky flow)、原鸡等亲缘关系次之,与猪(Sus scrofa)、绵羊(Ovis aries)等哺乳动物的亲缘关系最远。实时荧光定量PCR分析发现,MITF基因mRNA表达量以莆田黑鸭皮肤组织最高,极显著高于其他各组织(P<0.01);各组织依次为:皮肤>肾脏>肌胃>肝脏>肌肉。紫外分光光度计检测结果显示,莆田黑鸭各组织真黑色素的沉积规律与各组织MITF基因的表达量基本一致;其中肝脏组织MITF基因表达量与真黑色素含量呈显著正相关(P<0.05)。综上所述,MITF基因在莆田黑鸭中的表达具有组织特异性,可能对真黑色素的沉积具�Putian black duck （Anas anas domesticus） is the sole native variety in black-feather laying duck, which is rare waterfowl resource in China. The microphthalmia-associated transcription factor （MITF）, the most important transcription factor of melanocytes, could be induced by α-MSH （melanocyte-stimulating hormone） and bind to the cAMP responsive element binding protein （CREB） to activate MITF gene expression in melanocytes which plays a key role in the synthesis of melanin. To investigate the relationship between the expression of the MITF gene and regularity of eumelanin deposition in different tissues of Putian black duck, the MITF gene was firstly cloned and analyzed from the skin of Putian black duck with nested PCR and sequenced （GenBank No. MG516571）. MITF gene expression was detected in various tissues of Putian black ducks via real-time quantitative PCR, and deposition of eumelanin in the tissues was measured by UV spectrophotometry. The results showed that the length of MITF gene from Putian black duck was 1 341 bp, which contained the complete CDS sequence （12~1 334 bp） and encoded 440 amino acids. The amino acid homology compared with Anas platyrhynchos was 100%, and it was above 95% compared with chicken （Gallus gallus） and quail （Coturnix japonica）. The genetic relationship of MITF between Putian black duck and Anas platyrhynchos was the closest, followed with Anser cygnoides, Silky flow, Gallus gallus, and relationship compared with pig （Sus scrofa）, sheep （Ovis aries）, and other mammals was the farthest. The mRNA expression of MITF gene in Putian black duck skin tissue was the highest in the tissues （skin〉kidney〉gizzard〉liver〉muscle） （P〈0.01）. The deposition of eumelanin was basically consistent with the expression of MITF gene in each tissue of Putian black duck. The expression of MITF gene had significant positive correlation with the production of eumelanin in the liver tissues of Putian black duck （P〈0.05）. In summary, the expressi

关 键 词：莆田黑鸭 小眼畸形相关转录因子基因(MITF) 真黑色素 序列分析 基因表达 

分 类 号：S834.83[农业科学—畜牧学]