文章摘要
中华绒螯蟹组蛋白H2A基因的克隆及其在精子发生中的定位
Cloning and distribution of histone H2A during spermatogenesis of the Chinese mitten crab, Eriocheir sinensis
投稿时间:2019-03-13  修订日期:2019-05-28
DOI:
中文关键词: 中华绒螯蟹  H2A  基因克隆  精子发生
英文关键词: Eriocheir sinensis  histone H2A  gene cloning  spermatogenesis
基金项目:国家自然科学基金(31572269);国家自然科学( 31272309).,河北中医学院博士科研基金(BSZ2016003);
作者单位E-mail
吴江立 河北大学生命科学学院河北中医学院科技处 15200072669@126.com 
陈廷荣 河北大学生命科学学院  
穆淑梅 河北大学生命科学学院  
张晗 河北大学生命科学学院  
康现江 河北大学生命科学学院 xjkang@hbu.edu.cn 
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中文摘要:
      精子发生是指由精原细胞发育为成熟精子的过程。在大多数的物种中,这个过程涉及到与DNA结合的碱性蛋白的变化。体细胞类型的组蛋白全部或者部分被过渡蛋白替代,随后又被碱性更强的鱼精蛋白替代,伴随蛋白的逐级替换,此类动物精子核为浓缩的,染色质包装紧密。中华绒螯蟹(Eriocheir sinensis)的精子染色质呈松散的结构,其具体机制尚不明确。本研究利用PCR技术克隆了中华绒螯蟹组蛋白H2A基因的编码区,制备了其多克隆抗体,通过免疫荧光检测了组蛋白H2A在精子发生中的变化特征。结果显示,中华绒螯蟹组蛋白H2A基因编码区为369 bp,编码123个氨基酸,预测蛋白分子量为13.1 ku。氨基酸序列比对发现,H2A与南美白对虾(Penaeus vannmei)和斑节对虾(P. monodon)的同源性极高,均为99.19%。免疫荧光显示H2A存在于中华绒螯蟹精子发生的整个过程,在精原细胞和精母细胞,H2A在细胞核和细胞质均有表达,在精细胞和成熟的精子,H2A主要存在于细胞核。中华绒螯蟹成熟精子核内组蛋白H2A的保留可能与其非浓缩核有一定原因。研究结果为进一步探讨十足目甲壳动物非浓缩核机制提供了参考依据。
英文摘要:
      Spermatogenesis is the process by which spermatogonia develop into mature spermatozoa. In most animals, this process involves changes in the basic protein associated with DNA. Somatic-type histones are partially or totally replaced by transition proteins, which in turn are replaced by protamines with strong alkaline. With the replacement of basic proteins, nuclei progressively undergo chromatin condensation. The Chinese mitten crab (Eriocheir sinensis) sperm nucleus chromatin arrange loosely, its formation is not clear. In this research, DNA sequence encoding histone H2A was cloned by PCR method. Thereafter, polyclonal antibody against the histone H2A was generated in rabbit, histone H2A distribution during E. sinensis spermatogenesis was detected through immunofluorescence. The encoding sequence of E. sinensis H2A contains 369 bp that encodes a protein of 123 amino acids. The predicted molecular weight of H2A was 13.1 ku. Protein alignment studies demonstrated that this histone H2A had a high identity with its counterparts in Penaeus vannmei and P.monodon, and the homology both are 99.19%. Immunofluorescence demonstrated that histone H2A was present through the spermatogenesis of E.sinensis. In spermatogonia and spermatocytes, H2A was mainly distributed in the nuclei and cytoplasm, while in spermatids and spermatozoa, H2A was mainly in the nuclei. The persistence of H2A in the mature sperm nucleus of E. sinensis might have some association with the uncondensed chromatin. This work complements some extra data for further exploring the noncondensed sperm nuclei of decapod crustaceans.
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