Dmrt1基因敲除对罗非鱼精巢发育及减数分裂的影响.docVIP

  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*** 5 10 15 20 25 30 35 （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 nucleases，TALEN） 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（7、9、10 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：Q786 Dmrt1 knockout by TALENs on testicular development and meiosis in Nile tilapia, Oreochromis niloticus LI Minghui, YANG Huihui, LI Mengru, SUN Yunlv, WANG Tingru, SHI Hongjuan, WANG 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 factor involved in testicular differentiation and maintenance. In the present study, targeted gene disruption of Dmrt1 was performed in the Nile tilapia (Oreochromis niloticus) using TALENs. The efficiency was about 37%, including different bases deletions (7, 9, 10 etc). Histological observations of the 4-month-old Dmrt1 knock-out tilapia testis showed that it was developed abnormally with all germ cells being spermatogonia, with a few or even without any spermatocytes, spermatids and sperms, and without any seminiferous tubules. In addition, the central area of the testis was degraded to form a large cavity. Furthermore, some oocytes were appeared in the partially degraded area. Immunohistochemistry analysis showed that on the contrary to the control testis, aromatase, the key enzyme responsible for the syn