催乳素受体基因外显子10多态性及其与济宁青山羊高繁殖力关系的研究

Polymorphism of exon 10 of prolactin receptor gene and its relationship with prolificacy of Jining Grey goats

设计5对引物,采用PCR-SSCP技术检测催乳素受体(prolactin receptor,PRLR)基因外显子10及部分3′非翻译区在高繁殖力山羊品种(济宁青山羊)和低繁殖力山羊品种(辽宁绒山羊、波尔山羊和安哥拉山羊)中的单核苷酸多态性,同时研究该基因对济宁青山羊高繁殖力的影响。结果表明:首次拼接出的山羊PRLR基因外显子10及部分3′非翻译区的核苷酸序列长度为1,118bp,与已公布的绵羊、牛、人PRLR基因mRNA相应序列的同源性分别为98.33%、93.92%、74.52%,与已公布的羊驼PRLR基因部分序列的同源性为78.29%。引物P1、P2与P4扩增片段具有多态性,其余2对引物的扩增片段不存在多态性。对于P1扩增片段,在济宁青山羊和辽宁绒山羊中检测到AA型和AB型,在安哥拉山羊中检测到AA型和AC型,在波尔山羊中只检测到AA型;克隆测序表明AB型与AA型相比有两处突变(186G→A和220T→C),分别导致氨基酸由天冬氨酸变为天冬酰胺、亮氨酸变为脯氨酸;AC型与AA型相比有1处突变(140A→G),该突变没有导致氨基酸变化;济宁青山羊AA和AB基因型之间产羔数的最小二乘均值差异不显著(P>0.05)。对于P2扩增片段,在济宁青山羊、辽宁绒山羊和波尔山羊中都检测到DD型和DE型,而在安哥拉山羊中只检测到DD型;克隆测序表明DE型和DD型相比有两处突变(52G→A和122G→A),其中122bp处的突变导致氨基酸由精氨酸变为甘氨酸;济宁青山羊DD和DE基因型之间产羔数的最小二乘均值差异不显著(P>0.05)。对于P4扩增片段,在济宁青山羊中检测到FF型和FG型,在辽宁绒山羊中检测到FF型和GG型,在波尔山羊中只检测到FF型,在安哥拉山羊中检测到FF型、FG型和GG型;克隆测序表明GG型和FF型相比在扩增片段的143bp处发生1处碱基突变(A→G),并导致氨基酸由蛋氨酸变为缬氨酸;FG基因型济宁青山羊产羔数最小二乘均值比FF基因型的多0.76只(P<0.05)。研究结果初步表明:PRLR基因可能是控制济宁青山羊多胎性能的一个主效基因或是与之存在紧密遗传连锁的一个标记。

Prolactin receptor （PRLR） gene was studied as a candidate gene for the prolificacy of Jining Grey goats. Five pairs of primers were designed to detect single nucleotide polymorphisms of exon 10 and part of 3＇ untranslated region （UTR） of PRLR gene in both high fecundity breed （Jining Grey goat） and low fecundity breeds （Liaoning Cashmere goat, Boer goat and Angora goat） by PCR-SSCP. The nucleotide sequence of exon 10 and part of 3＇ UTR of caprine PRLR gene was spliced in this study for the first time. The length of this sequence was 1,118 bp. This sequence shared 98.33%, 93.92%, 74.52% homology with the published mRNA of PRLR gene of sheep, cow and human separately, and shared 78.29% ho- mology with the published partial genomic sequence of PRLR gene of the alpaca. Only the products amplified by primers P1, P2, P4 displayed polymorphisms. For primer P1, two genotypes （AA and AB） were detected in both Jining Grey goats and Liaoning Cashmere goats, two genotypes （AA and AC） were detected in Angora goats, and only genotype AA was detected in Boer goats. Sequencing revealed two mutations （186G→A and 220T→C） of PRLR gene in the genotype AB in comparison to the genotype AA. The former mutation resulted in an amino acid change of Asp→Asn, and the latter mutation resulted in an amino acid change of Leu→Pro. Only one mutation （140A→G） was found in the genotype AC in comparison to the genotype AA, and this mutation did not cause any amino acid change. The difference of the least squares means （LSM） for litter size between AA and AB was non-significant （P〉0.05） in Jining Grey goats. For primer P2, two genotypes （DD and DE） were detected in Jining Grey goats, Liaoning Cashmere goats and Boer goats, and only genotype DD was detected in Angora goats. Sequencing revealed two mutations （52G→A and 122G→A） of PRLR gene in the genotype DE in comparison to the genotype DD. The former mutation did not cause any amino acid change, and the latter mutation resulted in an amino acid change of Arg→Gly. The difference of LSM for litter size between DD and DE was non-significant （P〉0.05） in Jining Grey goats. For primer P4, two genotypes （FF and FG） were detected in Jining Grey goats, two genotypes （FF and GG） were detected in Liaoning Cashmere goats, only genotype FF was detected in Boer goats, and three genotypes （FF, FG and GG） were detected in Angora goats. Sequencing revealed one mutation （143A→G） of PRLR gene in the genotype GG in comparison to the genotype FF, and this mutation resulted in an amino acid change of Met→Val. The Jining Grey does with genotype FG had 0.76 （P〈0.05） kids more than those with genotype FF. These results preliminarily showed that the PRLR gene is either a major gene that influences the prolificacy of Jining Grey goats or a molecular marker in close linkage with such a gene.