大足黑山羊PHLDA2基因的克隆、组织表达与印记状况分析

doi:10.3864/j.issn.0578-1752.2015.02.14

摘要/Abstract

摘要： 【目的】克隆大足黑山羊PHLDA2（pleckstrin homology-like domain, family A, member 2）基因序列，分析其组织表达特征、印记状况以及与胎盘性状的关系，为深入研究该基因在山羊胎盘中的功能积累数据。【方法】根据人、牛和猪PHLDA2的保守区域设计引物以RT-PCR方法扩增大足黑山羊该基因部分cDNA序列，进一步利用5′-和3′-RACE技术获得全长cDNA序列;利用ORF Finder和ProtParam等在线工具分析PHLDA2基因序列特征;采用Real-time PCR方法分析PHLDA2在大足黑山羊心、肝、脾、肺、肾、大脑、肌肉、脂肪、舌和胎盘等10个组织的表达差异;寻找大足黑山羊和波尔山羊PHLDA2基因序列SNP位点，基于表达SNP的方法分析该基因在F₁代组织中的印记状况;采用线性回归的方法分析PHLDA2表达量与胎盘性状的关系。【结果】克隆得到935 bp大足黑山羊PHLDA2的cDNA全长序列,其中5′-UTR、3′-UTR和编码序列分别为62 bp、450 bp和420 bp，其编码140个氨基酸。预测的山羊PHLDA2蛋白分子量大小为15 638.7 Da，等电点为9.18，二级结构由α-螺旋(37.14%)、β-转角(9.29%)、延伸链(23.57%)和无规卷曲(30%)组成, 并且包含保守的PH（pleckstrin homology）结构域。③BLAST分析发现，山羊PHLDA2定位于29号染色体，其与牛、猪、猕猴、马和人同源基因核苷酸序列的相似性分别达到91%，90%，90%,90%和89%，在核苷酸和氨基酸序列上与牛的亲缘关系最近。④荧光定量PCR结果显示PHLDA2在山羊胎盘中表达量最高（P < 0.01)，在心、肝、肺、肾、肌肉、脂肪、舌和大脑组织中表达量很低且差异不显著（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。

关键词: 山羊, PHLDA2, 基因克隆, 荧光定量PCR, 组织表达, 印记分析

Abstract: 【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 < 0.01), with the highest mRNA level in the placenta, undetectable level in the spleen, and much less and no significant difference in the other 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 <0.01). 【Conclusion】 The sequence characteristics and imprinting status of PHLDA2 are conserved in goats, although it has a tissue-specific imprinting. The gene is an important candidate gene for goat placental growth and functions.

Key words: goat, PHLDA2, gene cloning, real-time PCR, tissue expression, imprinting analysis

苏鲁方，彭学强，蒋曹德. 大足黑山羊PHLDA2基因的克隆、组织表达与印记状况分析[J]. 中国农业科学, 2015, 48(2): 343-351.

SU Lu-fang, PENG Xue-qiang, JIANG Cao-de. Cloning, Tissue Expression and Imprinting Analysis of PHLDA2Gene in Dazu Black Goat[J]. Scientia Agricultura Sinica, 2015, 48(2): 343-351.

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