陈盼盼;郭巧生;史红专;郭丽媛;张会宁;
CHEN Pan-pan;GUO Qiao-sheng;SHI Hong-zhuan;GUO Li-yuan;ZHANG Hui-ning;Institute of Chinese Medicinal Materials, Nanjing Agricultural University;

• 1:南京农业大学中药材研究所

摘要(Abstract)：

蛋白激酶C(PKC)是一类广泛参与细胞增殖和发育的蛋白激酶,但在雌雄同体动物蚂蟥性腺中的发育机制尚不清楚。为探究蛋白激酶C在蚂蟥生殖细胞的发育过程中的作用,该研究克隆蚂蟥PKC基因,进行生物信息学分析,采用荧光定量PCR技术进行雌雄性腺的表达分析。结果表明:(1)克隆得到的Wp-PKC全长2 580 bp,相对分子质量76 555.19,包含1个编码670个氨基酸的开放阅读框,Wp-PKC和斑马鱼PKC-α及大鼠的PKC-γ亲缘关系较近,氨基酸序列相似度为55%和58%;(2)Wp-PKC编码的蛋白无信号肽,属于亲水性性蛋白,二级结构主要由无规则卷曲、α-螺旋、延伸链、折叠和组成,以无规则卷曲、α-螺旋占比最多,Wp-PKC蛋白不含有跨膜结构域,多序列比对和结构域预测分析显示,Wp-PKC包含4个经典蛋白激酶C的保守结构域;(3)荧光定量的结果显示Wp-PKC在蚂蟥性腺中的表达与生殖细胞的发育呈正相关,其中雄性腺中的表达显著高于雌性腺。综上,Wp-PKC为经典PKC,Wp-PKC可能对蚂蟥性腺发育尤其雄性腺发育起了促进作用,为蚂蟥性腺发育机制的进一步研究提供参考。
Protein kinase C(PKC) is a type of protein kinase widely involved in cell proliferation and development, but the developmental mechanism in the gonads of androgynous animals is still unclear. In order to explore the role of protein kinase C in the development of Whitmania pigra germ cells, the Wh. pigra PKC(Wp-PKC) gene was cloned, bioinformatics analysis was conducted, and fluorescent quantitative PCR was used to analyze the expression of female and male gonads. The results showed that:(1)The cloned Wp-PKC had a full length of 2 580 bp, a relative molecular weight of 76 555.19, and contains an open reading frame encoding 670 amino acids, Wp-PKC was closely related to Danio rerio PKC-α and rat PKC-γ. The similarity of amino acid sequence was 55% and 58%.(2)The protein encoded by Wp-PKC had no signal peptide and was a hydrophilic protein. The secondary structure is mainly composed of random coils, α-helices, extended chains, folds and folds, with the largest proportion of random coils and α-helices. Wp-PKC protein does not contain a transmembrane domain. Multiple sequence alignment and domain prediction analysis show that Wp-PKC contains 4 conserved domains of classical protein kinase C.(3)Fluorescence quantitative results showed that the expression of Wp-PKC in Wh. pigra gonads was positively correlated with the development of germ cells, and the expression in male gonads was significantly higher than that in female gonads. In summary, Wp-PKC is a classic PKC, and Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads, and provide references for further research on the developmental mechanisms of Wh. pigra.

关键词(KeyWords)： 蚂蟥;蛋白激酶C;雌雄性腺;克隆;表达分析
Whitmania pigra;protein kinase C;female and male gonads;cloning;expression analysis

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81673538,81473306)

作者(Author): 陈盼盼;郭巧生;史红专;郭丽媛;张会宁;
CHEN Pan-pan;GUO Qiao-sheng;SHI Hong-zhuan;GUO Li-yuan;ZHANG Hui-ning;Institute of Chinese Medicinal Materials, Nanjing Agricultural University;

Email:

DOI: 10.19540/j.cnki.cjcmm.20201114.102

参考文献(References)：

• [1] MUSASSHI M,OTA S,SHIROSHITA N.The role of protein kinase C isoforms in cell proliferation and apoptosis[J].Int J Hematol,2000,72(1):12.
• [2] GEIGESD,MEYER T,MARTE B,et al.Activation of protein kinase C subtypes alpha,gamma,delta,epsilon,zeta,and eta by tumor-promoting and nontumor-promoting agents[J].Biochem Pharmacol,1997,53(6):865.
• [3] O′BRIAN C,VOGEL V G,SINGLETARY S E,et al.Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue[J].Cancer Res,1989,49(12):3215.
• [4] 杨光明,徐竞,李涛,等.蛋白激酶Cε亚型在AVP调节休克血管MLC20磷酸化水平及MLCK/MLCP活性中的作用[J].中国现代应用药学,2009,26(9):712.
• [5] NANOS-WEBB A,BUI T,KARAKAS C,et al.PKCiota promotes ovarian tumor progression through deregulation of cyclin E[J].Oncogene,2015,35(19):2428.
• [6] XIAO H,LIU M.Atypical protein kinase C in cell motility[J].Cell Mol Life Sci,2013,70 (17):3057.
• [7] ARCHIBALD A,MAIA A M,ALYSON L S,et al.Atypical protein kinase C induces cell transformation by disrupting Hippo/Yap signaling[J].Mol Biol Cell,2015,26(20):3578.
• [8] SUN M,ZHANG H Y.Par3 and a PKC regulate BACE1 endosome-to-TGN trafficking through PACS1[J].Neurobiol Aging,2017,60:129.
• [9] SUGIOKA K,FIELMICH L E,MIZUMOTO K,et al.Tumor suppressor APC is an attenuator of spindle-pulling forces during C.elegans asymmetric cell division[J].Proc Natl Acad Sci USA,2018,115(5):E954.
• [10] 冯晨,袁莹,张杰,等.蛋白激酶C-δ在不同发育阶段小鼠睾丸的表达[J].解剖科学进展,2003,9(2):97.
• [11] 范衡宇,佟超,李世文,等.蛋白激酶C在小鼠卵母细胞体外成熟和受精中的作用[J].分子细胞生物学报,2003,36(1):37.
• [12] 罗雪梅,孟杰亲,陈秀芬,等.基于体外抗凝血实验研究铅污染对水蛭抗凝血作用的影响[J].中国中药杂志,2019,44(23):5114.
• [13] 史红专,刘宏,郭巧生,等.蚂蟥性腺及生殖细胞周年发育的组织学研究[J].中国中药杂志,2018,43(2):299.
• [14] AZZI A,BOSCOBOINIK D,HENSEY C.The protein kinase C family[J].EUR J Biochem,1992,208(3):547.
• [15] BOMFLEUR B,TAYLOR E L.Triassic leech cocoon from Antarctica contains fossil bell animal[J].Proc Natl Acad Sci USA,2012,109(51):20971.
• [16] ROTEM R,PAZ G F,HOMONNAI Z T,et al.Ca2+-independent induction of acrosome reaction by protein kinase C in human sperm[J].Endocrinology,1992(5):2235.
• [17] DOWNS S M,COTTOM J,HUNZICKER-DUNN M.Protein kinase C and meiotic regulation in isolated mouse oocytes[J].Mol Reprod Dev,2001,58(1):101.
• [18] KIM H M,SHIN T.Expression and localization of protein kinase C theta isoform in mouse testis[J].Life Sci,1999,65(8):805.
• [19] VIVEIROS M M,HIRAO Y,EPPIG J J.Evidence that protein kinase C (PKC) participates in the meiosis Ⅰ to meiosis Ⅱ transition in mouse oocytes[J].Dev Biol,2001,235(2):330.
• [20] COLONNA R,TATONE C,FRANCIONE A,et al.Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs[J].Mol Reprod Dev,1997,48(2):292.