The research was designed to analyze the linkage between the endogenous avian leucosis virus ev21 and phenotype of late feathering,as well as the promoter activity of LTR of ev21 gene.It was aimed to find the detection method for ev21 free breeding chickens.Long-range PCR was used to amplify the sequence of ev21 gene.The structure and its locus in chicken chromosome were analyzed based on the NCBI database.Two hundred and fifty-nine samples from 5 chicken lines were diagnosed for the detection of ev21.And 5'and 3'LTR were cloned into the pGL3-basic vector and were transiently transfected into A375 cells.The relative luciferase activity,which indicted the promoter activity of the transfected fragments,was detected.It was showed that the genome of ev21 long for 7 524 bp was successfully cloned and it was integrated between g.10681671-10681672 on chicken Z chromosome in reverse direction.Ev21 and late feathering were completely linked in Hy-line Gray,but were incompletely-linked in Taihang,Bashang and Hy-line Brown chicken.Especially,it was all positive in Hy-line Brown fast-feather rooster.5'LTR of ev21 showed a high promoter activity (P＜0.01),while 3'LTR did not show significantly higher activity than negative control (P＞0.05).These data showed that ev21 integration was not linked with late feathering in chickens.PCR for 7 590 bp is a method for screening of ev21 in chickens.5'LTR of ev21 had the high promoter activity.%旨在分析鸡内源ev21病毒与慢羽的连锁关系及其结构特征和启动子活性,为建立无内源ev21病毒的慢羽种鸡提供检测方法,并对ev21的启动转录活性进行探索.长片段PCR扩增获得鸡内源ev21病毒基因序列,检测太行鸡、坝上长尾鸡、海兰褐、海兰灰及其祖代五个品系259份样本的病毒携带情况.利用NCBI数据库Blast比对分析并PCR获得病毒基因全长.构建ev21基因5'和3'LTR区的pGL3-basic重组质粒转染A375细胞检测其启动子活性.结果显示:获得完整ev21病毒基因组全长7 524 bp,发现其反向插入在鸡Z染色体g.10681671-10681672之间,长片段7 590 bp PCR检测发现ev21与海兰灰慢羽鸡完全连锁,但与太行鸡、坝上长尾鸡和海兰褐慢羽并不完全连锁,尤其海兰褐快羽公鸡ev21全部阳性.ev21基因5'LTR区具有高强度的启动子活性(P＜0.01),3'LTR区活性高于阴性对照,但差异不显著(P＞0.05).综上,内源ev21病毒与鸡慢羽性状并不完全连锁,7 590 bp长片段PCR为内源ev21病毒的筛查提供检测方法,ev21基因5'LTR区具有强启动子活性.
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