奥利亚罗非鱼、尼罗罗非鱼MyoD1和MyoD2基因特征及差异

Characterization and difference analysis of MyoD1 and MyoD2 gene in Oreochromis aureus and Oreochromis niloticus

采用RT-PCR和RACE法,分离了奥利亚罗非鱼(Oreochromis aureus)、尼罗罗非鱼(O.niloticus)MyoD1和MyoD2基因全长cDNA。结果显示,2种罗非鱼MyoD1全长均为1090bp,包括5′非翻译区(UTR)137bp,3′UTR50bp,开放阅读框(ORF)903bp,编码300个氨基酸,其中第110～161个氨基酸为bHLH结构,第233～249个氨基酸为helixIII结构;MyoD2全长均为1478bp,包括5′UTR215bp,3′UTR471bp,ORF792bp,编码263个氨基酸,其中第91～142个氨基酸为bHLH结构,第212～228个氨基酸为helixIII结构。2种罗非鱼MyoD1与其他鱼类MyoD1的相似性为73%～92%;MyoD2与其他鱼类MyoD2的相似性为74%～79%。系统发育树显示,MyoD1和MyoD2分属两支,MyoD1所反映的不同鱼类间的亲缘关系符合传统分类。2种罗非鱼的MyoD1、MyoD2cDNA序列之间只存在个别碱基的差别,而氨基酸序列一致;奥利亚罗非鱼MyoD1的2个内含子均比尼罗罗非鱼的长。根据MyoD1内含子2的差异构建鉴别奥利亚罗非鱼和尼罗罗非鱼基因混杂的标记,对形态上典型的15尾奥利亚罗非鱼、18尾尼罗罗非鱼及15尾奥尼罗非鱼[Oreochromis aureus(♂)×Oreochromis niloticus(♀)]进行鉴定。结果其中1尾奥利亚罗非鱼中在MyoD1位点混杂了尼罗罗非鱼的基因,尼罗罗非鱼和奥尼罗非鱼则与预期的一致。该研究为选择基因纯合的奥利亚罗非鱼和尼罗罗非鱼提供了新的分子手段。

Myogenic Differentiation Antigen（MyoD）is a key member of myogenic regulatory factors（MRFs）protein family,which has a conserved basic helix-loop-helix（bHLH）domain.By using RT-PCR and RACE,MyoD1 and MyoD2 cDNA from Oreochromisaureus and O.niloticus were isolated.The results showed that MyoD1 cDN（A1090bp）of the two tilapias had the same 137bp 5＇-untranslated region（UTR）,50bp 3＇UTR and 903bp open reading frame（ORF）,which encoded a 350-amino-acid protein with a conserved bHLH domain（110-161 aa）and helix Ⅲ（233-249 aa）.MyoD2 cDNA（1478bp） of the two tilapias had the same 215bp 5＇UTR,471bp 3＇UTR and 792bp ORF,which encoded a 263-amino-acid protein with a conserved bHLH domain（91-142 aa）and helix Ⅲ（212-228 aa）.The similarity was 73%-92%between two kinds of tilapia＇s MyoD1 and the similarity was 74%-79%between tilapia＇s MyoD2 and other fishes＇MyoD2.The NJ phylogenetic tree of MyoD1 and MyoD2 indicated that all vertebrates＇MyoD1 and MyoD2 were clustered into two main branches,and fishes＇MyoD1 were basically consistent with the traditional classification.There was only some individual base differences in MyoD1 and MyoD2 cDNA between the two tilapias,and the amino acid sequence was identical to each other.The two introns of O.aureus MyoD1 were significant longer than those of O.niloticus MyoD1,while there was no obvious difference in two introns of MyoD2between two tilapias.According to the differences of MyoD1 intron 2,one molecular marker to distinguish O.aureus and O.niloticuswere established,and this marker was used to analyze the typical pure-blood 15 O.aureus,18 O.niloticusand and 15 O.aureus（♂）×O.niloticus（♀）selected by configuration.As a result,one in fifteen O.aureus was genetically mixed with O.niloticus at MyoD1 loci,and the result of 18 O.niloticus and 15 O.aureus（♂）×O.niloticus（♀） was the same as expected.The marker could serve as a molecular technique to select pure-blood individuals.