中华鳖组蛋白H2A变体克隆及其在卵母细胞中的表达分析  

CLONING AND EXPRESSION ANALYSIS OF HISTONE H2A VARIANT IN OOCYTES OF CHINESE SOFT-SHELLED TURTLE(PELODISCUS SINENSIS)

作　　者：祝骏贤 陈辰 刘晓莉 王亚坤[2] 雷骆 洪孝友[2] 于凌云[2] 徐红艳[2] 李伟[2] 朱新平[1,2] ZHU Jun-Xian;CHEN Chen;LIU Xiao-Li;WANG Ya-Kun;LEI Luo;HONG Xiao-You;YU Ling-Yun;XU Hong-Yan;LI Wei;ZHU Xin-Ping(Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214081,China;Key Laboratory of Tropical&Subtropical Fishery Resource Application&Cultivation of Ministry of Agriculture and Rural Affairs,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China)

机构地区：[1]南京农业大学无锡渔业学院,无锡214081 [2]中国水产科学研究院珠江水产研究所农业农村部热带亚热带水产资源利用与养殖重点实验室,广州510380

出　　处：《水生生物学报》2021年第6期1207-1213,共7页Acta Hydrobiologica Sinica

基　　金：国家重点研发计划(2018YFD0900200);广州市珠江科技新星(201806010131);广东省自然科学基金(2018A030313520);以农产品为单元的广东省现代农业产业技术体系创新团队建设项目(2019KJ150);中国-东盟海上合作基金(CAMC-2018F);中国水产科学研究院(珠江水产研究所)科技创新团队(2020TD35和2020ZJTD01)资助。

摘　　要：为研究组蛋白H2A对龟鳖动物生殖细胞发育分化作用和机制,克隆了中华鳖(Pelodiscus sinensis)组蛋白H2A变体的同源物(命名为PsH2A),分析其转录本的表达模式及在卵巢发育成熟过程中的细胞定位。PsH2A cDNA序列全长575 bp,5′端非编码区68 bp,3′端非编码区108 bp,开放阅读框399 bp,编码133个氨基酸。氨基酸序列比对结果显示其与龟类的H2A变体同源性更高,与哺乳类同源性较低。RT-qPCR和RT-PCR结果显示,PsH2A转录本在1冬龄、2冬龄和3冬龄的中华鳖卵巢中高表达(P<0.01),而在精巢和其他成体组织中几乎检测不到。化学原位杂交结果显示,PsH2A mRNA在卵母细胞中特异性表达,其中初级卵母细胞中表达信号最强,且均匀的分布在细胞质中。随着卵母细胞发育成熟进入到生长期和成熟期后,目的信号逐渐减弱,并且主要在核周区域表达。此外,PsH2A mRNA的相对表达量也表现出中华鳖卵巢发育的季节性变化。综上,研究结果表明PsH2A在中华鳖卵母细胞发育过程中可能发挥着重要作用。Histone H2A and its variants play an important role in many cellular processes.However,their role in the germ cells differentiation of turtle has not been well characterized yet.Here,we cloned the Chinese soft-shell turtle(Pelodiscus sinensis)PsH2A and analyzed its expression pattern and cellular location during oogenesis.The 575 bp PsH2A cDNA was isolated from P.sinensis,which contains an open reading frame(ORF)of 399 bp encoding a protein of 133 amino acid residues,a partial 5′untranslated region(5′-UTR)of 68 bp and a partial 3′untranslated region(3′-UTR)of 108 bp.Real-time quantitative PCR(RT-qPCR)result showed that PsH2A mRNA expression level was higher in ovary than other tissues(P<0.01),such as heart,liver,spleen,kidney,brain,muscle,and testis.Reverse transcription PCR(RT-PCR)result showed that PsH2A mRNA only detected in ovary.Moreover,in the male and female gonads of 1-year,2-year and 3-year-old P.sinensis,the PsH2A mRNA was only significantly expressed in ovary(P<0.01).The results of chemical in situ hybridization(CISH)showed that PsH2A mRNA was specifically expressed in oocytes.When primary oocytes developed to stageⅢ,strong signal could be detected and evenly distributed in the cytoplasm.From growing follicular stage(stageⅦoocytes)to mature follicular stage(stageⅨoocytes),the PsH2A mRNA signal gradually weakened and spread in the cytoplasm around the nucleus.The signal was distributed in the perinuclear cytoplasm,but was undetected in the peripheral cytoplasm.Furthermore,the relative expression level of PsH2A mRNA also responded to seasonal change in ovarian development in P.sinensis.In 1-year-old and 3-year-old ovaries,the relative level of PsH2A mRNA increased as ovary developing.However,in 2-year-old ovary,the relative level of PsH2A mRNA increased first and then decreased,reaching a peak level in April.In addition,in different season,the relative expression of PsH2A mRNA showed a downward trend with age increasing.In summary,these findings suggest that PsH2A may play an important role

关 键 词：中华鳖 组蛋白H2A变体 卵母细胞 季节表达 细胞定位 

分 类 号：Q344.1[生物学—遗传学]