鸡ev21占位区和未占位区重复序列结构与羽速表型关系研究

Relation between Feather Speed and Gene Structure of Repeated Sequences of ev21 Occupied-site and Unoccupied-site Regions in Chicken

本试验旨在探讨鸡羽速基因型Hae Ⅲ酶切鉴定方法的分子基础。以羽型明确的6个品系(太行鸡、坝上长尾鸡、大午粉鸡、海兰灰鸡、海兰褐鸡、海兰灰祖代四系鸡)313份鸡基因组为模板,利用Hae Ⅲ酶切的RFLP试验进行羽速验证,并对ev21占位区(OS)和非占位区(US)共有序列以及OS区特有序列进行酶切试验。结果表明:1)Blast分析发现1 450bp为鸡ev21的US区域片段。海兰灰及其祖代四系和大午粉祖代的羽速基因型与HaeⅢ酶切结果完全一致,但太行慢羽公鸡、慢羽母鸡、坝上长尾慢羽公鸡和海兰褐慢羽鸡的一致率分别为40.0%、27.6%、28.6%和0.0%;2)太行鸡和坝上长尾鸡ev21的OS和US区共有序列538bp酶切鉴定结果与表型一致率达到92%以上,其他品系(除海兰褐快羽公鸡为0.0%)均为100.0%;3)鸡ev21的OS区特异序列1 440bp的扩增阳性率在太行慢羽公鸡、慢羽母鸡和快羽公鸡中的阳性率分别为94.1%、65.5%和0.0%,在海兰灰祖代鸡中分别为100.0%和0.0%,并且1 440片段不能被Hae Ⅲ切开。综合分析6个品系ev21的OS和US区结构特征,认为US区Hae Ⅲ酶切位点变异不能作为慢羽和内源病毒ev21的鉴定依据,而OS区的ev21插入与1 440bp序列Hae Ⅲ酶切位点的A→G突变和八碱基重复是紧密连锁的。

The aim of this study was to explore the molecular basis of Hae Ⅲ digestion detection of genotypes of chicken feather speed in chicken. Three hundred and thirteen individuals in 6 strains of chickens （Taihang, Bashang Long-tail, Dawufen, Hy-line Gray, Hy-line Brown, pro- genitor of Hy-line Gray） with definite phenotype of feather speed were detected with RFLP. The com- mon region of occupied site（OS） and unoccupied site（US） for 538 bu, also the suecific region of OS for 1 440 bp, were digested with Hae Ⅲ. The results showed that： 1） The fragment for 1 450 bp was the unoccupied-site repeated sequence for ev21 ascertained by Blast. The digestion detection results of Hae Ill for 1 450 bp were completely consistent with the phenotype of HY-line Gray and progenitors of HY-line Gray and Dawufen, but their consistency with Taihang slow-feather cock, Taihang slow-feather hen, Bashang Long-tail slow-feather cock and HY-line Brown slow-feather chicken were 40. 0%, 27.6%, 28.6% and 0.0%, respectively. 2） The consistency was above 920% between digestion results of 538 bp and phenotype in Taihang and Bashang Long-tail chicken, and the other strains were 100.0% except for HY-line Brown fast-feather cock with 0.0%. 3） The positive rate of 1 440 bp fragment PCR in slow-feather Taihang cock, Taihang hen and fast-feather Taihang cock was 94. 1%, 65. 5% and 0.0%, respectively, and it was 100.0K and 0.0% in slow-feather and fast-feather progenitors of Hy- line gray, respectively. The 1 440 bp fragment could not be digested by Hae Ⅲ and it could be a detec- tion basis for ev21. The structure was analyzed comprehensively for the OS and US of ev21 in 6 strains of chicken and it was concluded that the site recognized by Hae Ⅲ enzyme in US could not be a detection base for slow-feather and ev21, while ev21 insertion in OS and the site for Hae Ⅲ digestion in 1 440 bp were linked with the mutation of A→G and 8 bases repeated.