三个品种家鸡催乳素基因cDNA的克隆及序列分析  

Cloning and Sequencing of Prolactin Gene cDNA in Three Chicken Breeds

作　　者：周敏[1] 张细权[1] 施振旦[1] 曹永长[1] 

机构地区：[1]华南农业大学动物科学系,广州510642

出　　处：《Acta Genetica Sinica》2001年第7期614-620,共7页

基　　金：国家自然科学基金项目(39700102);广东省自然科学基金项目(970012);国家重点基础研究发展计划项目(G20000

摘　　要：从粤黄鸡、丝毛乌骨鸡及伊莎蛋鸡的垂体中快速抽提总ＲＮＡ，根据国外已发表的肉用仔鸡催乳素基因ｃＤＮＡ的序列，设计并合成了能与特定载体末端互补的１对引物，经反转录－聚合酶链式反应（ＲＴ－ＰＣＲ）方法扩增获得了特异性片段。将扩增片段与线性化质粒 ｐＢＳＳＫ连接，克隆后进行序列分析，与已报道的肉用仔鸡、矮脚鸡和火鸡催乳素基因的核苷酸序列及推导的氨基酸序列进行了比较。结果表明，不同品种间核苷酸同源性介于９３．９７％～９９．８７％之间，其中丝毛乌骨鸡与矮脚鸡间的同源性最高，为９９．８７％。推导的相应氨基酸序列的同源性在９８．２５％～１００％之间，也是丝毛乌骨鸡与矮脚鸡间的同源性最高，为１００％。在粤黄鸡、丝毛乌骨鸡和伊莎蛋鸡中，发现前两者的催乳素前体的ｃＤＮＡ片段推导的氨基酸序列中信号肽裂解位点跟肉用仔鸡、矮脚鸡及火鸡的一样，为 Ｌｅｕ－Ｐｒｏ－Ｉｌｅ－Ｃｙｓ，而伊莎蛋鸡的信号肽裂解位点则不一样，为Ｐｒｏ－Ｐｒｏ－Ｉｌｅ－Ｃｙｓ。此位点的差异可能导致催乳素前体翻译加工的不同，使伊莎蛋鸡无就巢性。这３个家鸡品种与国外的肉用仔鸡、矮脚鸡催乳素氨基酸序列还在以下位置出现差异：７１、１４１、１５０、１７５。在肉用仔鸡、丝毛乌骨鸡?The total RNA was extracted from the pituitary of two Chinese native chicken breeds, Yuehuang and Taihe Silkies, and one layer Isa, using RNeasy Kit (QIAGEN, Germany). The total RNA was used to synthesize a specific fragment with RT-PCR, in which the primers were designed based on the sequence of broiler prolactin gene cDNA. The specific fragment was ligated to a linear plasmid PBSSK and cloned with XL1-Blue. The sequencing of prolastin cDNA was carried out with ABI PRISHTM 377XL DNA Sequencer after cloning. The cDNA sequences and deduced amino acid sequences of prolactin gene of two Chinese native chicken breeds and one layer were compared with that of broiler, dwarf chicken and turkey. There was 93.97%-99.89% cDNA sequence homology among Chinese native, layer, broiler and dwarf chickens, in which there was the highest (99.87%) between those of Taihe and dwarf chickens. There was 98.25%- 100% deduced amino acid sequence homology among Chinese native, layer, broiler and dwarf chickens, in which there was the highest (100%) between those of Taihe Silkies and dwarf chickens. It was found that Yuehuang and Taihe Silkies had the same signal peptide cleavage site Leu-Pro-Ile-Cys among amino acids sequence deduced from pre-prolactin cDNA as broiler, dwarf and turkey, while layer Isa had a different cleavage silo Pro-Pro-Ile-Cys. Such difference might cause a different translation processing of pre-prolactin, which could make layer Isa non-broody. There were different amino acids in the positions 71, 141, 150 and 175 of deduced prolactin amino acid sequences among Yuehuang, Taihe Silkies, layer, broiler and dwarf chickens. There was a heparin-binding site in positions 175- 181 (L-R-R-D-S -H-K) among prolactin amino acid sequences of broiler and Taihe Silkies.

关 键 词：家鸡 催乳素 基因克隆 序列分析 CDNA 

分 类 号：S831[农业科学—畜牧学]