大足黑山羊 PHLDA2基因的克隆、组织表达与印记状况分析

摘要

[Objective]The objectives of this study are to clone goat PHLDA2 (pleckstrin homology-like domain, family A, member 2), to analyze its tissue expression and imprinting status, and to reveal its relationship with placental traits, thereby providing data for further investigation on the gene functions in goat placenta.[Method]Partial cDNA sequence of PHLDA2 was cloned for Dazu black goats according to the conserved regions among human, cattle and pig sequences, and the full-length cDNA of this gene was further cloned with 5′- and 3′-RACE. Sequence characteristics of goat PHLDA2 were analyzed using online softwares, such as ORF Finder and ProtParam. Real-time PCR was applied to examine tissue expression of goat PHLDA2 in 10 tissues, including heart, liver, spleen, kidney, skeletal muscle, lung, tongue, fat, brain and placenta. Imprinting status of the gene in placenta was investigated by expressed SNP method. And, linear regression estimation was used to reveal the correlation between PHLDA2 expression and placental traits.[Result]A full-length cDNA of 935 bp was cloned for goat PHLDA2. The cDNA contains 62 bp 5′-UTR, 450 bp 3′-UTR and 420 bp coding sequence, and encodes a protein of 140 amino acids in total. The molecular weight of goat PHLDA2 protein is 15638.7 Da, and its theoretical isoelectric point is 9.18. The secondary structure of the protein contains α-helix (37.14%),β-turn (9.29%), extended strand (23.57%) and random coil (30%). The result of BLAST showed that goat PHLDA2 gene was located on goat chromosome 29, and that it had high sequence similarity with the homologues of cattle (91%),pig (90%), macaque (90%), horse (90%) and human(89%). Furthermore, goat PHLDA2 has the closest phylogenetic relationship to the cattle gene in both nucleotide and amino acids sequences. Different expressions of goat PHLDA2 were detected among tissues (P 0.05). Imprinting analysis demonstrated that this gene was expressed from the maternal allele in goat placenta, heart and liver tissues, but biallelically expressed in the lung, brain and skeletal muscle. In addition, the expression levels of goat PHLDA2 were significantly correlated with placental weight (R2=0.855. P 0.05),在脾中未检测到表达。⑤印记分析表明，PHLDA2在初生山羊胎盘、心脏和肝脏组织表达母源等位基因，但在肺、大脑和肌肉组织为双等位基因表达。⑥PHLDA2表达水平与山羊胎盘重回归关系显著（n=15，R2=0.855，P <0.01）。【结论】山羊 PHLDA2基因序列和结构特征具有物种间的保守性，但是其印记状况具有组织特异性； PHLDA2为山羊胎盘的生长和功能调控的重要基因。