Abstract:Full length cDNA sequence of calcineurin B homologous protein 2 (CHP2) gene as a key molecule involved in regulation of intracellular pH and cell proliferation was amplified in black porgy Acanthopagrus schlegelii (AsCHP2) by RACE PCR, and the sequence characteristics and homology of the AsCHP2 were analyzed by bioinformatics. The expression of AsCHP2 mRNA was detected in whole black porgy larvae, and brain, heart, muscle, liver and gonads of 90 d or 180 d old black porgy hybrid F2 (red sea bream Pagrus major ♂×black porgy ♀) and, backcross F1 (black porgy♂×hybrid F1♀) by real-time fluorescent quantitative PCR. It was found that the AsCHP2 cDNA was 1722 bp in length, including a 69 bp 5′-untranslated region (UTR), a 1069 bp 3′-UTR and a 585 bp open reading frame encoding a peptide of 194 amino acids. The protein contained two conserved EF-hand domains and a nuclear export sequence, the molecular weight of about 21. 87 ku, pI of about 4. 89. Secondary and three-dimensional structure of AsCHP2 was developed by homology modeling. Nuclear sequence alignment revealed that AsCHP2 shared higher identities (86%—81%) with other fishes, and the alignment of the predicted proteins sequence of AsCHP2 had 80%—72% of similarity with other fishes. The evolutionary relationships of AsCHP2 amino acid sequences with other kinds were analyzed using the methods of NJ tree and ML tree. Sequence alignment and structure comparison revealed that AsCHP2 were members of the conservative CHP2. The AsCHP2 appeared to be constitutively expressed in early larval development (5—35 d) and the 90-day and 180-day old individuals, and had high expression level in all tissues from the individuals of 90-day old. CHP2 mRNAs was ubiquitously expressed in all tested tissues, and far more abundant in the heart, as about several times as that in gonad in black porgy, the hybrid F2 and the backcross F1. These findings suggest that the CHP2 gene might be involved in the growth and development of sea fishes.
[1]张洪菊,李庆华,王立洪,等.EF-hand对钙调磷酸酶B同源蛋白2的生物学功能影响[J].中国医药导报,2012,9(35):28-31. [2]李彬,李洪强,马丽,等.肿瘤相关基因chp2在白血病细胞中表达的研究[J].中国实验血液学杂志,2008,16(4):734-737. [3]Hammam A A, Eissa H H, El Masry M R, et al. CHP2 gene expression and quantitation in Egyptian patients with acute leukemia[J].Meta Gene,2014,2:323-331. [4]Ammar Y B, Takeda S, Hisamitsu T, et al. Crystal structure of CHP2 complexed with NHE1-cytosolic region and an implication for pH regulation[J].The EMBO Journal,2006,25(11):2315-2325. [5]Li Q H, Wang L H, Lin Y N, et al. Nuclear accumulation of calcineurin B homologous protein 2 (CHP2) results in enhanced proliferation of tumor cells[J].Genes to Cells,2011,16(4):416-426. [6]李庆华,庞天翔,袁文肃,等.在血清饥饿条件下CHP2调节NHE1活性减少细胞死亡[J].细胞生物学杂志,2005,27(5):573-577. [7]张剑,龚昆梅,赵泉,等.敲降钙调磷酸酶B同源蛋白2表达导致静脉血管内皮细胞增殖迁移抑制和细胞周期停滞的研究[J].中国血管外科杂志(电子版),2018,10(2):133-137. [8]Zaun H C, Shrier A, Orlowski J. Calcineurin B homologous protein 3 promotes the biosynthetic maturation,cell surface stability,and optimal transport of the Na+/H+ exchanger NHE1 isoform[J].The Journal of Biological Chemistry,2008,283(18):12456-12467. [9]Di Sole F, Vadnagara K, Moe O W, et al. Calcineurin homologous protein: a multifunctional Ca2+-binding protein family[J]. American Journal of Physiology and Renal Physiology,2012,303(2):165-179. [10]Fray E, Wallert M, Provost J. Binding of calcineurin homologous protein isoforms 1 and 2 for the Na+/H+ exchanger isoform 1 using recombinant fluorescent proteins[J]. Faseb Journal,2015,29(1):45-64. [11]蔺亚妮,李庆华,王立洪,等.钙调磷酸酶B同源蛋白2核定位对宫颈癌HeLa细胞增殖能力的影响[J].中华临床医师杂志(电子版),2012,6(24):8022-8027. [12]李国栋,王月丹,陈慰峰.CHP与钙调神经磷酸酶的活性调节[J].自然科学进展,2004,14(8):852-855. [13]Jean C T, Lee S C, Hul C F, et al. Genetic variation of black porgy, Acanthopagrus schlegeli, (Perciformes: Sparidae) in the coastal waters of Taiwan [J]. Biochemical Systematics and Ecology,1996,24(3):211-219. [14]单保党,洪万树,何大仁.真鲷与黑鲷杂交与多倍体育种系列研究Ⅰ[J].海洋渔业,1999,21(1):14-15. [15]Park I S, Kim B S, Lee S J, et al. Comparative morphometric traits of hybrids between red sea bream (Pagrus major) and black sea bream (Acanthopagrus schlegelii)[J]. Fisheries Science and Technology,2006,9(1):44-47. [16]陈淑吟,张志勇,刘海林,等.黑鲷×真鲷杂交子代回交繁育及子代早期发育观察[J].水产科学,2017,36(6):693-699. [17]许晨璐,孙晓梅,张守攻.基因差异表达与杂种优势形成机制探讨[J].遗传,2013,35(6):714-726. [18]Bendtsen J D, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides: Signal P 3.0 [J]. Journal of Molecular Biology,2004,340(4):783-795. [19]Nielsen H, Krogh A. Prediction of signal peptides and signal anchors by a hidden Markov model[J].Proceedings: International Conference on Intelligent Systems for Molecular Biology,1998,6:122-130. [20]Schwede T, Kopp J, Guex N, et al. SWISS-MODEL:an automated protein homology-modeling server[J].Nucleic Acids Research,2003,31(13):3381-3385. [21]Altschul S F, Madden T L, Schäffer A A, et al. Gapped BLAST and PSI-BLAST:a new generation of protein database search programs[J].Nucleic Acids Research,1997,25(17):3389-3402. [22]Thompson J D, Gibson T J, Plewniak F, et al. The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,1997,25(24):4876-4882. [23]Kumar S, Stecher G, Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1870-1874. [24]Pang T X, Su X H, Wakabayashi S, et al. Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers[J].The Journal of Biological Chemistry,2001,276(20):17367-17372. [25]Nagita M, Inoue H, Nakamura N, et al. Two nuclear export signals specify the cytoplasmic localization of calcineurin B homologous protein 1[J].Journal of Biochemistry,2003,134(6):919-925. [26]吁亭,赵宇威,余绍宁. EF-Hand蛋白研究进展[J]. 生物物理学,2013,1(1):1-10. [27]王立洪.肿瘤标志物中性粒细胞明胶酶相关载脂蛋白(NGAL)与钙调磷酸酶B同源蛋白2(CHP2)的生物学功能研究[D].北京:北京协和医学院,2011. [28]Li G D, Zhang X, Li R, et al. CHP2 activates the calcineurin/nuclear factor of activated T cells signaling pathway and enhances the oncogenic potential of HEK293 cells [J]. Journal of Biological Chemistry,2008,283(47):32660-32668. [29]Inoue H, Nakamura Y, Nagita M, et al. Calcineurin homologous protein isoform 2 (CHP2), Na+/H+ exchangers-binding protein, is expressed in intestinal epithelium[J]. Biological & Pharmaceutical Bulletin,2003,26(2):148-155. [30]陈淑吟,李鹏,张志勇,等.黑鲷♀×真鲷♂反交子代与黑鲷的CaM基因克隆与mRNA表达分析[J].中国水产科学,2017,24(6):1193-1202. [31]钱晓萍,孙秀媛,李国栋,等.肿瘤相关抗原CHP2促进HEK293细胞在裸鼠腹腔内的转移[J].北京大学学报(医学版),2009,41(5):521-524. [32]曲迅,杨美香,刘福利,等.肿瘤相关抗原编码基因CHP2对细胞增殖的抑制作用[J].中国肿瘤生物治疗杂志,2003,10(4):253-256. [33]杨美香,曲迅,韩克军,等.生物信息学在新基因HCA520研究中的作用[J].药物生物技术,2004,11(4):216-220. [34]雷冠雄.钠氢离子交换蛋白(NHE)调节因子CHP1和CHP2的表达研究[D].天津:南开大学,2007.