鲤鱼基因组修饰和ENU诱变技术的建立及其在成骨细胞与肌肉纤维发育研究中的应用

发布时间：2018-01-07 18:33

本文关键词：鲤鱼基因组修饰和ENU诱变技术的建立及其在成骨细胞与肌肉纤维发育研究中的应用　出处：《苏州大学》2016年博士论文　论文类型：学位论文

【摘要】：鲤鱼作为一种重要的经济鱼类,其年产量大约为300万吨,占世界上整个淡水经济鱼类年产量的10%。鲤鱼还作为一种脊椎动物模型,在生态学、演化生物学、环境毒理学、生理学、营养学、免疫学、发育生物学、遗传育种及转基因等多方面的研究中得以广泛应用。但是,由于其肌间刺较多,阻碍了鲤鱼成为世界范围的食材。因此,降低鲤鱼肌间刺的数量,提高鲤鱼品质及经济价值成为亟需解决的问题之一。鲤鱼是二倍化的四倍体物种,且繁殖周期长,传统的育种和遗传研究具有较大的困难。TALEN和CRISPR-Cas9技术是最近几年兴起的基因修饰技术,使得快速靶向修饰基因成为可能。本研究中,我们采用了TALEN和CRISPR-Cas9技术对鲤鱼的硬骨发育系列关键基因,包括sp7、runx2、bmp2a、spp1和opg,以及肌肉抑制因子mstn进行修饰,以期得到稳定遗传的理想品系。利用系统树分析发现鲤鱼有两个sp7基因,四个mstn基因,而斑马鱼仅有一个sp7和两个mstn,符合鲤鱼基因组为二倍化四倍体的特点。首先,本研究利用TALEN方法成功的对鲤鱼sp7、runx2、spp1和mstn中的靶序列诱导了突变,并且有较高的突变效率;然后利用CRISPR-Cas9技术分别成功地修饰了鲤鱼的两个sp7基因,sp7a和sp7b,结果发现两个基因的突变均造成了严重的骨骼缺陷。Micro-CT结果表明鲤鱼sp7a和sp7b突变体的颅面骨和椎体骨发育均显著慢于对照组,但是sp7a-CRISPR突变鲤鱼比sp7b-CRISPR的突变体有更严重的骨骼发育缺陷。茜素红染色结果表明sp7a-CRISPR突变体相对于对照组,表现出明显的骨骼缺陷,包括鳃盖和上颌骨的发育不全、血管脊椎不规则、椎体畸形以及肌间刺的长度较短等。而mstnba突变的鲤鱼则在肌肉细胞的数量和肌肉纤维面积上相对于对照组出现了显著增加,由此导致体重和体长显著高于对照组。统计分析显示mstnba-CRISPR组内突变体的体重和突变率呈明显的正相关性。Western印迹表明CRISPR-Cas9介导的突变破坏了鲤鱼骨骼肌的Mstn信号通路。qRT-PCR与HE染色结果共同表明mstnba-CRISPR突变的鲤鱼F0代的肌肉纤维既表现出增生,也表现出肥大。我们还利用CRISPR-Cas9技术成功的构建了sp7a;mstnba鲤鱼双突变体,并且两个基因的突变率均很高。这些研究表明了TALEN和CRISPR-Cas9技术均能够高效的修饰鲤鱼的基因组,为鲤鱼遗传研究和育种提供了新的途径。另外,ENU诱变是一种可以改良农业物种遗传性状的有用方法。我们使用ENU处理鲤鱼的成熟精子,然后体外受精得到ENU处理的F1代鲤鱼。结果表明高浓度的ENU造成鲤鱼胚胎畸形较多。使用合适浓度的ENU处理得到的F1代幼鱼及成鱼的体重分布范围较大,并且其中大约16%的成年鲤鱼表现出明显的骨骼缺陷。这些结果表明ENU处理成熟精子也是一种改良鲤鱼遗传育种的有效方法。
[Abstract]:Carp as an important economic fish, its annual production is about 3 million tons, accounting for the world's total freshwater economic fish production of 10 percent. Carp also as a vertebrate model, in ecology. Evolutionary Biology, Environmental Toxicology, Physiology, Nutrition, Immunology, Developmental Biology, genetic breeding and Transgene have been widely used. Therefore, reducing the number of carp muscle spines and improving the quality and economic value of carp become one of the problems that need to be solved. Carp is a diploid tetraploid species. The breeding cycle is long and the traditional breeding and genetic research is more difficult. Taren and CRISPR-Cas9 are the most popular gene modification techniques in recent years. In this study, we used TALEN and CRISPR-Cas9 techniques to identify a series of key genes, including sp7, for the development of carps. Runx2, bmp2a, spp1 and OPG, as well as muscle suppressor mstn, were modified. Using phylogenetic analysis, it was found that carp had two sp7 genes and four mstn genes, while zebrafish had only one sp7 and two mstn. First of all, the TALEN method was used to induce the mutation of the target sequences of carp sp7, runx2, spp1 and mstn. And has higher mutation efficiency; Then, two sp7 genes, sp7a and sp7b, were successfully modified by CRISPR-Cas9 technique. The results showed that both mutations caused serious bone defects. Micro-CT results showed that the craniofacial and vertebral bone development of carp sp7a and sp7b mutants was significantly slower than that of the control group. But sp7a-CRISPR mutant carp had more serious skeletal defects than sp7b-CRISPR mutants. Alizarin red staining showed that sp7a-CRISPR mutants were relative to each other. In control group. Demonstrated significant bone defects, including dysplasia of the branchial and maxillary bones, and irregular vascular vertebrae. The number of muscle cells and muscle fiber area of mstnba mutant carp increased significantly compared with the control group. As a result, the body weight and body length were significantly higher than those of the control group. Statistical analysis showed that there was a significant positive correlation between body weight and mutation rate in the mstnba-CRISPR group. Western blot showed CR. ISPR-Cas9 mediated mutation destroyed the Mstn signaling pathway of carp skeletal muscle. QRT-PCR and HE staining results showed that mstnba-CRISPR mutant carp F0. The muscle fibers of the generation both show hyperplasia. We also use CRISPR-Cas9 technology to successfully construct sp7a; Mstnba carp double mutants, and the mutation rate of the two genes are very high. These studies show that both TALEN and CRISPR-Cas9 can efficiently modify the genome of carp. It provides a new way for the genetic research and breeding of carp. In addition, mutagenesis is a useful method to improve the genetic traits of agricultural species. We use ENU to treat the mature sperm of carp. After in vitro fertilization, F1 carp treated with ENU were obtained. The results showed that high concentration of ENU resulted in more malformation of carp embryo. The body weight distribution of F1 generation juvenile and adult fish treated with appropriate concentration of ENU was higher. It's a big area. And about 16% of the adult carp showed obvious skeletal defects. These results showed that ENU treatment of mature sperm is also an effective method to improve carp genetics and breeding.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q953

【相似文献】