香猪雌激素受体α、β基因cDNA的克隆及分析

Identification and characterization of cDNA encoding estrogen receptor alpha and beta in Xiang pigs

本文采用RT-PCR技术,分别从香猪的子宫和卵巢总RNA中扩增了雌激素受体α和β(ERα、ERβ)两种cDNA,分别长1788bp和1581bp,包括起始密码子和终止密码子,碱基序列与大白猪的ERα、ERβ基因的相似性为99.3%和99.6%。ERα、ERβ两个基因编码595、526个氨基酸,N-末端的20、24个氨基酸残基为信号肽,成熟肽序列与大白猪之间均有4个氨基酸不同。三维结构分析发现,与大白猪相比,香猪ERα成熟肽第192、231位氨基酸由Ser、Met变为Gly、Thr,位于DNA结合结构域,成熟肽438位氨基酸由Val变为Gly,位于ERα的配体结合结构域;ERβ成熟肽中,167位和360位氨基酸由Asp和Phe变为Glu和Pro,位于受体的DNA结合域和配体结合域,这五个位点的氨基酸替代可能影响ERs蛋白与雌激素受体应答元件、雌激素等配体的结合,改变相关基因的转录效率,并可能影响香猪的卵巢、子宫等繁殖系统的发育,与香猪的低繁殖力有关。

Estrogen receptors （ERs） belong to the steroid/thyroid hormone receptor superfamily of nuclear receptors which upon binding of ligand are activated and bind to specific DNA response elements to initiate the transcription of target genes. Both estrogen receptor alpha （ERα） and estrogen receptor beta （ERβ） are responsible for the fertility of pig breeds. To elucidate the cause of low litter size of Xiang pigs, a local breed in Guizhou Province, the complete cDNA fragments encoding ERa and ERβ were isolated from the ovary and uterus by reverse transcription polymerase chain reaction （RT-PCR）. The complete open reading frame （ORF） of ERa and ERβ contained 1788 bp and 1581 bp respectively with 99.3% and 99.6% nucleotide identity respectively to the Large white breed. The deduced amino acids of ERα and ERβ of Xiang pig were 595 and 526 residues with 20 or 24 amino acid residues at the N-terminus of ERα and ERβ recognized to be signal peptides. Compared with large white pig, four amino acid substitutions were found in the mature peptides of ERα and ERβ which were G192S, T231M, G438V and V568A in ERα and K99R, E167D, P360F and P496S in ERβ. In the eight substitutions, amino acid residues at 192 and 231 in ERα and 167 in ERβ were located at the DNA-binding domain, amino acid residues at 438 in ERα and 360 in ERβ involved at the ligand- binding domain. These substitutions at DNA-binding and ligand-binding domains might affect both of ER-DNA interactions and ER-protein interactions and the affinity of tight nuclear binding between the ERs-estrogen complex and estrogen response element （ERE） in Xiang pigs. These results suggested that the five substitutions at ERα and ERβ might contribute to the low fecundity of Xiang pigs and may benefit our understanding of the mechanisms controlling fertility in this pig breed