半滑舌鳎基因组编辑TALEN技术的建立及雄性决定基因dmrt1的功能分析

发布时间：2018-04-29 06:33

本文选题：半滑舌鳎 + 显微注射技术　；参考：《上海海洋大学》2017年博士论文

【摘要】：一、半滑舌鳎基因组编辑TALEN技术的建立基因组编辑技术对于海洋动物基因功能分析和基因工程育种具有重大意义和应用价值,开展海水养殖动物基因组编辑技术的研究,对于培育定点突变的海水养殖动物新品种至关重要。但由于海水养殖鱼类胚胎显微注射技术难度大,至今少有成功的报道,加之海水鱼类仔鱼数量过少、难以培育成活等问题,严重阻碍了海水鱼类基因组编辑技术的研究进程,迄今国内外未见有关海水养殖鱼类基因组编辑的成功报道。本研究成功建立了海水鲆鲽鱼类半滑舌鳎胚胎显微注射技术及基因组编辑技术,并在性别决定基因功能验证上取得突破性进展。将GFP质粒成功显微注射到半滑舌鳎胚胎中并表达;利用TALEN技术成功敲除了半滑舌鳎Z染色体上的dmrt1基因,并获得了33尾基因突变鱼。二、半滑舌鳎dmrt1基因在性别决定及性腺发育过程中的功能本研究通过靶向dmrt1基因的TALEN m RNA显微注射到半滑舌鳎胚胎中,成功诱导dmrt1基因的突变,突变效率最高达100%。ZZ型dmrt1突变鱼发育成中间有一个腔的卵巢样精巢,精子发生被阻断。Dmrt1突变雄鱼的性腺中雌性性别相关基因foxl2和cyp19a1a显著性增加。Dmrt1突变雄鱼比正常雄鱼生长快,与正常雌鱼长势一样。同时,检测到1尾雌雄同体鱼,该鱼的遗传性别为ZW型,其上侧性腺外形上像精巢,下侧性腺外形上像卵巢。上下侧性腺切片染色结果显示,上侧性腺呈现典型的精巢结构,而下侧性腺则呈现卵巢样结构。采用PCR分析了雌雄同体鱼性腺的dmrt1基因突变情况,在上侧的精巢中未检测到的dmrt1基因的突变,而在下侧的卵巢样性腺中则检测到dmrt1基因的许多突变。由此表明dmrt1基因是半滑舌鳎雄性精巢发育必不可少的基因,是半滑舌鳎的雄性决定基因,并且是关联着生长二态性。三、半滑舌鳎雄性性别决定基因dmrt1突变家系的建立通过基因组编辑技术敲除了半滑舌鳎dmrt1基因,将dmrt1基因定点突变鱼培育至性成熟。然后分别在2015年和2016年通过人工繁育技术将筛选出的dmrt1基因定点突变的F0代ZZ雄鱼与野生型雌鱼、突变的F0代ZW雌鱼与野生型雄鱼、突变的F0代雌鱼与雄鱼分别进行杂交得到杂交子代鱼,并进行了子代鱼苗的dmrt1基因的检测。结果证明,父母本的dmrt1突变基因遗传到了子代鱼中。未来还需要做进一步的人工选育工作,并最终获得生长快的家系。
[Abstract]:Establishment of Genome Editing TALEN technique for Cynoglossus semilaevis Genome Editing technique has great significance and application value for gene function analysis and genetic engineering breeding of marine animals. It is very important to cultivate new species of mariculture animals with site-directed mutagenesis. However, due to the difficulty of microinjection of marine fish embryos, there are few successful reports so far, in addition to the small number of marine fish larvae, it is difficult to grow and survive, which seriously hinders the research process of marine fish genome editing technology. So far, there has been no successful report on genome editing of mariculture fish at home and abroad. In this study, the embryo microinjection and genome editing techniques of sole sole were successfully established, and a breakthrough was made in the functional verification of sex-determining genes. The GFP plasmid was microinjected into the embryo of Cynoglossus semilaevis and the dmrt1 gene on Z chromosome of Cynoglossus semilaevis was successfully knocked out by TALEN technique and 33 mutant fish were obtained. Second, the function of dmrt1 gene in sex determination and gonadal development of Cynoglossus semilaevis. The aim of this study was to induce the mutation of dmrt1 gene by microinjection of TALEN m RNA targeting dmrt1 gene into the embryo of Cynoglossus semilaevis. The mutational efficiency of 100%.ZZ type dmrt1 mutant fish developed into an ovary like nest with a middle cavity. The sex-related genes foxl2 and cyp19a1a in the gonad of the male with blocked spermatogenesis. Dmrt1 mutated male grew faster than the normal male, and the female sex related genes foxl2 and cyp19a1a increased significantly in the gonadal gland of the male. It's the same as the normal female. At the same time, a hermaphrodite fish was detected. Its genetic character was ZW type. The shape of the upper gonad was similar to that of the testis, and the shape of the lower gonad looked like the ovary. The results of upper and lower gonad sections showed that the upper gonad presented a typical spermary structure, while the lower gonad presented an ovarian like structure. PCR was used to analyze the mutation of dmrt1 gene in the gonad of the hermaphrodite fish. The mutation of dmrt1 gene was not detected in the upper testis, but many mutations of the dmrt1 gene were detected in the ovary like gland on the lower side. It is concluded that dmrt1 gene is essential for the development of male spermatozoa of Cynoglossus semilaevis and is the male determinant gene of Cynoglossus semilaevis and is related to growth dimorphism. Third, the establishment of the dmrt1 mutation family of male sex determinant gene of Cynoglossus semilaevis by genomic editing technique, the dmrt1 gene of Cynoglossus semilaevis was knocked out, and the dmrt1 gene site-directed mutant fish was bred to sexual maturity. The selected F0 generation ZZ male and wild female, the mutant F0 generation ZW female and the wild male were selected by artificial breeding in 2015 and 2016, respectively. The F _ 0 generation female and male fish were hybridized to obtain the hybrid offspring, and the dmrt1 gene of the offspring was detected. The results showed that the parent's dmrt1 mutation gene was inherited into the offspring of fish. In the future, further artificial breeding is needed, and fast growing families will eventually be obtained.
【学位授予单位】：上海海洋大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4

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