猪JHDM2A基因的克隆及表达分析

doi:10.16431/j.cnki.1671-7236.2017.06.004

摘要/Abstract

摘要：

试验旨在克隆猪JHDM2A基因，并研究其在猪卵巢组织中的表达情况。首先克隆猪JHDM2A基因，并构建pLVX-IRES-ZsGreen1-JHDM2A真核表达载体，同时对JHDM2A基因在猪卵泡发育过程中的表达情况进行分析。结果显示，克隆得到的猪JHDM2A基因编码区长度为3 945 bp，编码1 315个氨基酸。通过多重氨基酸序列比对发现，猪JHDM2A基因与黄牛、水牛、绵羊和人相应氨基酸序列的同源性分别为93.5%、94.7%、94.7%和93.8%。蛋白质分子系统进化树分析结果表明，JHDM2A基因在物种进化过程中高度保守。通过脂质体转染法将构建的pLVX-IRES-ZsGreen1-JHDM2A真核表达载体导入HEK293T细胞，可观察到清晰的绿色荧光蛋白表达。免疫组化结果显示，JHDM2A蛋白在不同发育阶段猪卵泡中均有表达。本试验通过克隆获得猪JHDM2A基因序列，JHDM2A蛋白在猪卵巢中高度表达，表明其功能可能与猪卵泡发育密切相关。

关键词: 猪; JHDM2A基因; 克隆; 卵泡; 表达分析

Abstract:

The aim of this study was to clone the JHDM2A gene of porcine and study the expression of JHDM2A gene in porcine ovary. In this study, we cloned the porcine JHDM2A gene and constructed its eukaryotic expression vector, the expression of JHDM2A gene in porcine ovarian tissue was also analyzed. The results showed that the cloned CDS length of porcine JHDM2A gene was 3 945 bp, which encoded 1 315 amino acids. The results of multiple amino acid sequence comparison showed that the porcine JHDM2A shared 93.5%, 94.7%, 94.7% and 93.8% homologous compared with those of Bubalus bubalis, Bos taurus, Ovis aries and Homo sapiens, respectively. Phylogenetic tree analysis indicated that the JHDM2A gene was highly conservative in the evolutionary process. The pLVX-IRES-ZsGreen1-JHDM2A eukaryotic expression vector was constructed, and clear green fluorescent signal was observed when the plasmid was transfected into HEK293T cells by liposome method. The immunohistochemical results showed that the JHDM2A protein was expressed in porcine follicle of different development stages.The results showed that the porcine JHDM2A gene sequences was cloned, and the JHDM2A protein was highly expressed in porcine ovary, indicating that its function might be closely related to the development of porcine follicular.

Key words: porcine; JHDM2A gene; cloning; follicle; expression analysis

中图分类号:

Q785

宋延霞, 吴大平, 李海艳, 李胜, 粟小平, 李湘萍. 猪JHDM2A基因的克隆及表达分析[J]. 《中国畜牧兽医》, 2017, 44(6): 1596-1603.

SONG Yan-xia, WU Da-ping, LI Hai-yan, LI Sheng, SU Xiao-ping, LI Xiang-ping. Cloning and Expression Analysis of Porcine JHDM2A Gene[J]. , 2017, 44(6): 1596-1603.

参考文献

[1] Tsukada Y, Fang J, Erdjument-Bromage H, et al. Histone demethylation by a family of JmjC domain-containing proteins[J]. Nature, 2006, 439(7078):811-816.
[2] Martin C, Zhang Y. The diverse functions of histone lysine methylation[J]. Nature Reviews Molecular Cell Biology, 2005, 6(11):838-849.
[3] 冯兵,朱莹,贺嵩敏,等. 罗勒多糖对缺氧条件下肝癌细胞组蛋白H3K9me2甲基化及G9a、JMJD1A表达的影响[J]. 中药材, 2015, 38(7): 1460-1465.
[4] Cho H S, Toyokawa G, Daigo Y, et al. The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G[J]. International Journal of Cancer, 2012, 131(3):E179-E189.
[5] Klose R J, Yamane K, Bae Y, et al. The transcriptional repressor JHDM3A demethylatestrimethyl histone H3 lysine 9 and lysine 36[J]. Nature, 2006, 442(7100):312-316.
[6] Garciabassets I, Kwon Y S, Telese F, et al. Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors[J]. Cell, 2007, 128(3):505-518.
[7] Pedanou V E, Stéphane G, Sébastien T, et al. The histone H3K9 demethylase KDM3A promotes anoikis by transcriptionally activating pro-apoptotic genes BNIP3 and BNIP3L[J]. Elife, 2016, 5(2016):e16844-e16860.
[8] 彭文培,曾长军. 精子发生及冷冻过程中表观遗传调控的研究进展[J]. 中国畜牧兽医, 2014, 41(9): 199-205.
[9] Okada Y, Scott G, Ray M K, et al. Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis[J]. Nature, 2007, 450(7166):119-123.
[10] Javadirad S M, Hojati Z, Ghaedi K, et al. Expression ratio of histone demethylase KDM3A to protamine-1 mRNA is predictive of successful testicular sperm extraction in men with obstructive and non-obstructive azoospermia[J]. Andrology, 2016, 4(3):492-499.
[11] Tateishi K, Okada Y, Kallin E M, et al. Role of Jhdm2a in regulating metabolic gene expression and obesity resistance[J]. Nature, 2009, 458(7239):757-761.
[12] Ko S Y, Kang H Y, Lee H S, et al. Identification of Jmjd1a as a STAT3 downstream gene in mES cells[J]. Cell Structure & Function, 2006, 31(2):53-62.
[13] Ma D K, Chiang C H, Ponnusamy K, et al. G9a and Jhdm2a regulate embryonic stem cell fusion-induced reprogramming of adult neural stem cells[J]. Stem Cells, 2008, 26(8):2131-2141.
[14] Beyer S, Kristensen M M, Jensen K S, et al. The histone demethylases JMJD1A and JMJD2B are transcriptional targets of hypoxia-inducible factor HIF[J]. Journal of Biological Chemistry, 2008, 283(52):36542-36552.
[15] Chakraborty D, Wei C, Rosario G X, et al. HIF-KDM3A-MMP12 regulatory circuit ensures trophoblast plasticity and placental adaptations to hypoxia[J]. Proc Natl Acad Sci USA, 2016,113(46):E7212-E7221.
[16] Fan L, Peng G, Sahgal N, et al. Regulation of c-Myc expression by the histone demethylase JMJD1A is essential for prostate cancer cell growth and survival[J]. Oncogene, 2015, 75(15 Supplement):2441-2452.
[17] Pa M, Naizaer G, Seyiti A, et al. KDM3A confers metastasis and chemoresistance in epithelial ovarian cancer[J]. Journal of Molecular Histology, 2016, 46(2):511-518.
[18] Justin N, De M V, Aasland R, et al. Reading, writing and editing methylated lysines on histone tails: New insights from recent structural studies[J]. Current Opinion in Structural Biology, 2010, 20(6):730-738.
[19] Knebel J, Haro L D, Janknecht R. Repression of transcription by TSGA/Jmjd1a, a novel interaction partner of the ETS protein ER71[J]. Journal of Cellular Biochemistry, 2006, 99(1):319-329.
[20] Yang H, Liu Z, Yuan C, et al. Elevated JMJD1A is a novel predictor for prognosis and a potential therapeutic target for gastric cancer[J]. International Journal of Clinical & Experimental Pathology, 2015, 8(9):11092-11099.
[21] Park S Y, Park J W, Chun Y S. Jumonji histone demethylases as emerging therapeutic targets[J]. Pharmacological Research, 2016, 105:146-151.
[22] Parrish J K, Sechler M, Winn R A, et al. The histone demethylase KDM3A is a microRNA-22-regulated tumor promoter in Ewing Sarcoma[J]. Oncogene, 2015, 34(2):257-262.
[23] Hoghoughi N, Hojati Z. Study of the relation between rs11677451 and rs 2030259 polymorphic markers in Jhdm2a gene and male infertility[J]. Cell Journal (Yakhteh), 2013, 15(Sup 1):45-46.
[24] Yamane K, Toumazou C, Tsukada Y, et al. JHDM2A, a JmjC-Containing H3K9 demethylase, facilitates transcription activation by androgen receptor[J]. Cell, 2006, 125(3):483-495.
[25] Suzuki T, Miyata N. Lysine demethylases inhibitors[J]. Journal of Medicinal Chemistry, 2011, 54(24):8236-8250.
[26] Nomura N, Nagase T, Miyajima N, et al. Prediction of the coding sequences of unidentified human genes[J]. OSA Publishing, 1995, 54(35):10443-10453.