Dmrt基因敲除对罗非鱼精巢发育及减数分裂的影响.doc

Dmrt1基因敲除对罗非鱼精巢发育及减数分裂的影响 Dmrt1 基因敲除对罗非鱼精巢发育及减数分裂的影响李明辉杨惠惠李梦茹孙运侣王婷茹石红娟王德寿5101520253035西南大学生命科学学院淡水鱼类资源与生殖发育教育部重点实验室重庆 400175摘要转录激活因子样效应物核酸酶Transcription activator-like effector nucleasesTALEN是目前最有发展前景的基因组修饰技术Dmrt1 是脊椎动物精巢分化和维持的关键基因本文在养殖鱼类尼罗罗非鱼Oreochromis niloticus建立了 TALEN 靶向基因敲除技术通过对 Dmrt1 基因敲除结果发现TALEN 能高效约 37的对 Dmrt1 基因进行编辑包括删除不同碱基数目7910 等的突变类型4 月龄 Dmrt1 敲除个体性腺组织学观察显示精巢不能正常发育如不能正常起始减数分裂因此少有甚至没有对照组精巢中所见的精母细胞精细胞和精子仅见大量精原细胞部分退化区域甚至出现卵母细胞同时未见曲细精管免疫组化结果显示 Dmrt1 敲除个体精巢中检测到芳香化酶合成雌激素的关建酶的表达而没有检测到胆固醇侧链裂解酶 P450scc 的表达以上结果表明像哺乳动物一样Dmrt1 在硬骨鱼类精巢的正常分化发育以及精原细胞减数分裂起始中起重要作用关键词水生生物学TALEN基因敲除Dmrt1减数分裂罗非鱼中图分类号Q786Dmrt1 knockout by TALENs on testicular development andmeiosis in Nile tilapia Oreochromis niloticusLI Minghui YANG Huihui LI Mengru SUN Yunlv WANG Tingru SHI HongjuanWANG Deshou Key Laboratory of Freshwater Fish Reproduction and Development Ministry of Education School of Life Science Southwest University ChongQing 400715 Abstract Recent studies have demonstrated that transcription activator-like effector nuclease TALEN is the most promising genome modification technology Dmrt1 is the key factorinvolved in testicular differentiation and maintenance In the present study targeted genedisruption of Dmrt1 was performed in the Nile tilapia Oreochromis niloticus using TALENsThe efficiency was about 37 including different bases deletions 7 9 10 etc Histologicalobservations of the 4-month-old Dmrt1 knock-out tilapia testis showed that it was developedabnormally with all germ cells being spermatogonia with a few or even without anyspermatocytes spermatids and sperms and without any seminiferous tubules In addition thecentral area of the testis was degraded to form a large cavity Furthermore some oocytes wereappeared in the partially degraded area Immunohistochemistry analysis showed that on thecontrary to the control testis aromatase the key enzyme responsible for the synthesis of estrogenwas observed in the Dmrt1 knockout testis while P450scc expres