达氏鲟dmc1基因的克隆及在精子生成中的表达分析  

Cloning and expression characterization of dmc1 gene during spermatogenesis in Acipenser dabryanus

作　　者：向浩 叶欢[2] 李创举[2] 杨俊琳 厉萍[2] 杜浩[2] 危起伟[2] XIANG Hao;YE Huan;LI Chuang-ju;YANG Jun-lin;LI Ping;DU Hao;WEI Qi-wei(College of Fisheries, Huazhong Agricultural University, Wuhan430070, China;Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan430223, China;Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan430072, China)

机构地区：[1]华中农业大学水产学院,湖北武汉430070 [2]中国水产科学研究院长江水产研究所,农业部淡水生物多样性保护重点实验室,湖北武汉430223 [3]中国科学院水生生物研究所,湖北武汉430072

出　　处：《海洋渔业》2019年第4期434-443,共10页Marine Fisheries

基　　金：中央级公益性科研院所基本科研业务费-水产生物生殖调控相关机制及技术研究-达氏鲟精原干细胞体外增殖与冷冻保存(2015B02YQ01);中国水产科学研究院基本科研业务费(2018JBF10);国家自然科学基金面上项目(No.31772854);中央公益性基本科研业务费项目(2017HY-ZD0405);973项目-中华鲟性腺发育关键营养物质体内转化的生理基础(2015CB150702)

摘　　要：DNA减数分裂重组酶1基因(disrupted meiotic cDNA,dmc1)是一个在减数分裂时期特异表达的基因,为减数分裂同源染色体配对所必需。为研究dmc1基因在达氏鲟(Acipenser dabryanus)不同组织的表达特征,从达氏鲟性腺转录组数据库搜索得到该基因并设计引物,克隆获得达氏鲟dmc1的编码区序列,其中开放阅读框(ORF)为1 029 bp,编码342个氨基酸。运用生物信息学方法分析达氏鲟dmc1基因编码的蛋白序列和系统进化关系,推测达氏鲟Dmc1蛋白的分子量为82.696 kDa,理论等电点(PI)为5.04;多重序列比对发现,Dmc1氨氮酸序列在进化中可能比较保守。系统进化分析显示,AdDmc1属于RecA家族Dmc1蛋白分支,且与四足动物类聚为一支。RT-PCR结果表明,dmc1基因在精巢和卵巢中特异表达。结合组织学结果通过实时荧光定量PCR研究dmc1基因在精子生成过程中的表达特征发现,dmc1基因在0~Ⅰ期精巢中不表达;在Ⅱ、Ⅲ、Ⅳ期精巢中,其表达量逐渐升高并在Ⅳ期精巢中达到最高;在Ⅴ期精巢中,其表达量显著性下降(P<0.05),因此推断达氏鲟dmc1基因可能是减数分裂时期特异表达的基因,研究结果可为后续达氏鲟初级和次级精母细胞的鉴定奠定基础。Disrupted meiotic cDNA(dmc1) is a meiotic-specific gene that encodes a protein required for homologous chromosome synapsis during the prophase of meiosis Ⅰ. To determine the expression characterization of Dabry’s sturgeon Acipenser dabryanus dmc1 gene in different tissues, the partial cDNA sequence of Addmc1 was obtained from gonadal transcriptome database, and then the coding sequence of Addmc1 gene was cloned, which contained an ORF of 1 029 bp encoding a protein of 342 amino acids. The predicted protein secquence and phylogenetic relationships were analyzed by bioinformatics methods. It showed that the predicted sequence had a molecular mass of 82.696 kDa and a pI of 5.04. A comparison of the deduced amino acid sequence of Addmc1 with that of other fishes and tetrapods was made, showing that the Dmc1 shared high identity of more than 87% among any species, and they mainly contained two motifs as GEFRTGKT and LLIID that were combined with nucleic acid, indicating that Addmc1 may be highly conserved during the evolution. A phylogenetic analysis showed that the clone isolated in this study encoded a A.Dabryanus homolog of Dmc1 and clustered with the tetrapod. RT-PCR analysis revealed that Addmc1 was only detected in the testis and ovary, not in other examined tissues, including heart, liver, spleen, kidney, gill, intestines, pituitary and hypothalamus. The HE staining results of testes in 1-3 years old male A.Dabryanus were as follows: in the stage of 0, the testis had not formed the seminiferous lobule, a few number of primordial germ cells could be found. When testis stayed in the stage of Ⅰ, a large number of type A spermatogonia could be observed as in the stage of spermatogenesis proliferation. With the differentiation of spermatogonia, we could find some type B spermatogonia, but type A spermatogonia occupied most in the stage of II, whereas in the stage of III, the testis entered into the meiosis, most cells differentiated into primary spermatocytes, a few number of secondary spermatocyte could be fo

关 键 词：达氏鲟 dmc1 减数分裂 性腺发育 表达分析 

分 类 号：Q786[生物学—分子生物学]