猪Cdk2基因的克隆及其功能研究

doi:10.11843/j.issn.0366-6964.2013.10.003

摘要/Abstract

摘要：

本研究旨在克隆猪Cdk2基因，并研究其编码蛋白CDK2的生物学功能。采用RT-PCR扩增猪Cdk2基因，运用生物信息学软件分析其核苷酸和编码氨基酸特征，并预测编码蛋白的生物学功能；利用半定量RT-PCR方法分析该基因在猪各个脏器和组织中的表达情况；共聚焦显微镜观察CDK2的亚细胞定位，采用过表达和shRNA干扰技术研究CDK2在细胞周期和细胞增殖中的调控作用。结果表明，猪Cdk2基因开放阅读框(ORF)为897 bp（GenBank：JX967576），该基因与绵羊、牛、山羊、人、金仓鼠、小鼠、仓鼠和沟鼠Cdk2的核苷酸相似性依次为94.2%、94.0%、93.8%、93.4%、91.8%、91.0%、90.6%和 89.9%，与牛、山羊和绵羊的亲缘关系最近；Cdk2编码298 aa，CDK2分子质量为34 ku。Cdk2 mRNA在猪10个不同脏器和组织中均有表达。CDK2定位于细胞质和细胞核中，并通过蛋白酶体途径降解。猪CDK2在PK-15细胞中过表达引起S期细胞比例显著减少及G2/M期细胞比例显著增加（P<0.05），而G0/G1期无显著变化；相反，CDK2表达量降低引起S期细胞比例显著减少及G0/G1期细胞比例显著增加，而G2/M期无显著变化。本研究成功克隆了猪Cdk2基因并对其编码蛋白生物学功能进行了初步研究。

Abstract:

This study was aimed to clone the porcine cyclin-dependent kinase 2 (Cdk2) gene and investigate the function of CDK2 encoded by Cdk2. In this study, porcine Cdk2 cDNA was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). Bioinformatic approaches were used to analyze the nucleotide and amimo acid sequences as well as the bio-function of CDK2. Semi-quantitative RT-PCR was used to analyze the tissue expression profile of Cdk2 mRNA, confocal microscopy was used to observe the subcellular localization of CDK2. Overexpression and shRNA techniques were both employed to investigate the role of CDK2 in cell cycle and cell proliferation regulation. Results showed that Cdk2 gene (GenBank accession no. JX967576) contained an 897 bp open reading frame and shared 94.2%, 94.0%, 93.8%, 93.4%, 91.8%, 91.0%, 90.6% and 89.9% identity with those of sheep, cattle, goat, human, golden hamster, house mouse, Chinese hamster and norway rat, respectively. Porcine Cdk2 gene coded 298 aa, with a molecular weight of 34 ku. The mRNA transcripts of porcine Cdk2 were found in 10 different porcine tissues by semi-quantative RT-PCR. Porcine CDK2 was localized in both the nucleus and cytoplasm and degradated through proteasomal pathway. Porcine CDK2 overexpression induced a significant decrease in proportion of cell at S-phase and G2/M-phase arrest, but no changes at G0/G1-phase. In contrast, when the CDK2 expression was down-regulated by shRNA, a significant decrease in proportion of cell at S-phase and G0/G1-phase arrest was observed, without changes at G2/M-phase. The porcine Cdk2 gene was successfully cloned and its function was also investigated.

中图分类号:

S828
S813.3

唐青海，张辉，危艳武，刘长明. 猪Cdk2基因的克隆及其功能研究[J]. 畜牧兽医学报, 2013, 44(10): 1522-1531.

TANG Qing-hai, ZHANG Hui, WEI Yan-wu, LIU Chang-ming. Cloning and Functional Analysis of Porcine Cdk2 Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(10): 1522-1531.

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