南方鲇scp3和dmc1基因的克隆、组织分布及体外相互作用  

Cloning,Tissue Distribution and In vitro Interaction of scp3 and dmc1 Genes in Silurus meridionalis

作　　者：刘枝华 董然然 钱佳铭 张显波 LIU Zhi-Hua;DONG Ran-Ran;QIAN Jia-Ming;ZHANG Xian-Bo(College of Animal Science/Institute of Special Aquatic Products,Guizhou University,Guiyang 550025,China;Fishery Research Institute,GuizhouAcademy ofAgricultural Sciences,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院/特种水产研究所,贵阳550025 [2]贵州省农业科学院水产研究所,贵阳550025

出　　处：《农业生物技术学报》2021年第8期1574-1583,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31802270);贵州省科技厅基础研究一般项目(黔科合基础[2019]1112号);贵州省科技计划项目(黔科合平台人才[2018]5781号);贵州大学培育项目(黔科合平台人才[2017]5788-18)。

摘　　要：联会复合体3(synaptonemal complex 3,SCP3)和DNA减数分裂重组酶1(DNA meiotic recombinase 1,DMC1)是减数分裂过程中的关键因子,南方鲇(Silurus meridionalis)中未见相关报道。本研究利用PCR、荧光免疫组化和Western blot技术,对scp3和dmc1进行克隆、亚细胞定位及组织表达分析,通过GST pull-down实验验证体外相互作用。结果显示,南方鲇scp3(GenBank No.MW075231)和dmc1(GenBank No.MF770581)的CDS为723和1029 bp,分别编码240和342个氨基酸;同源性分析表明,南方鲇SCP3和DMC1与黄颡鱼(Tachysurus fulvidraco)的同源性分别高达89.17%和97%;系统进化分析中,南方鲇SCP3和DMC1与其他鱼类聚成一支,然后与四足类聚成一支;反转录PCR(reverse transcription-PCR,RT-PCR)结果显示,在南方鲇的9个组织中,scp3和dmc1仅在卵巢中表达;qRT-PCR结果显示,scp3和dmc1在55 dah(days after hatching)卵巢中的表达量最高,显著高于30和90 dah的表达量;荧光免疫组化的结果显示,SCP3在南方鲇卵母细胞的细胞核和细胞质中有较强的阳性信号,DMC1在卵母细胞的细胞核中有较强的阳性信号;GST pull-down结果显示,SCP3和DMC1存在体外相互作用。本研究结果为深入探讨南方鲇减数分裂过程中SCP3和DMC1作用机制提供基础资料,为人工繁殖南方鲇获得高质量卵子提供参考依据。Synaptonemal complex 3(SCP3)and DNA meiotic recombinase 1(DMC1)are key factors in meiosis,which have not been reported in Silurus meridionalis.In present study,PCR,fluorescence immunohistochemistry and Western blot were used to clone and analyze the sub-cellular distribution and tissue expression of scp3 and dmc1;GST pull-down was used to verify in vitro protein-protein interaction.The results showed that the CDS of scp3(GenBank No.MW075231)and dmc1(GenBank No.MF770581)were 723 and 1029 bp,encoding 240 and 342 amino acids,respectively.Homology analysis showed that the homology of S.meridionalis SCP3 and DMC1 to Tachysurus fulvidraco were as high as 89.17%and 97%,respectively.In phylogenetic analysis,S.meridionalis SCP3 and DMC1 were clustered with fish,and then clustered with tetrapod.Reverse transcription-PCR(RT-PCR)results showed that scp3 and dmc1 were only specifically expressed in ovary.The results of qRT-PCR showed that relative expression of scp3 and dmc1 had the highest level in 55 dah(days after hatching),which were significantly higher than that of 30 and 90 dah.The results of fluorescence immunohistochemistry showed strong positive signals of SCP3 in nucleus and cytoplasm of oocytes,and strong positive signal of DMC1 in the nucleus of oocytes.GST pull-down assay showed that SCP3 interacted with DMC1 in vitro.This study could provide basic data for further study on the function of SCP3 and DMC1 during meiosis,and reference for artificial breeding of S.meridionalis to obtain high-quality eggs.

关 键 词：DNA减数分裂重组酶1(DMC1) 联会复合体3(SCP3) 南方鲇 基因表达 荧光免疫组化 GST pull-down 

分 类 号：S961.21[农业科学—水产养殖]