發(fā)布時間：2018-01-07 18:33

  本文關鍵詞：鯉魚基因組修飾和ENU誘變技術的建立及其在成骨細胞與肌肉纖維發(fā)育研究中的應用　出處：《蘇州大學》2016年博士論文　論文類型：學位論文

  更多相關文章： 鯉魚 CRISPR-Cas9 TALEN ENU-mutagenesis sp7 mstn 肌間刺

【摘要】：鯉魚作為一種重要的經濟魚類,其年產量大約為300萬噸,占世界上整個淡水經濟魚類年產量的10%。鯉魚還作為一種脊椎動物模型,在生態(tài)學、演化生物學、環(huán)境毒理學、生理學、營養(yǎng)學、免疫學、發(fā)育生物學、遺傳育種及轉基因等多方面的研究中得以廣泛應用。但是,由于其肌間刺較多,阻礙了鯉魚成為世界范圍的食材。因此,降低鯉魚肌間刺的數量,提高鯉魚品質及經濟價值成為亟需解決的問題之一。鯉魚是二倍化的四倍體物種,且繁殖周期長,傳統(tǒng)的育種和遺傳研究具有較大的困難。TALEN和CRISPR-Cas9技術是最近幾年興起的基因修飾技術,使得快速靶向修飾基因成為可能。本研究中,我們采用了TALEN和CRISPR-Cas9技術對鯉魚的硬骨發(fā)育系列關鍵基因,包括sp7、runx2、bmp2a、spp1和opg,以及肌肉抑制因子mstn進行修飾,以期得到穩(wěn)定遺傳的理想品系。利用系統(tǒng)樹分析發(fā)現鯉魚有兩個sp7基因,四個mstn基因,而斑馬魚僅有一個sp7和兩個mstn,符合鯉魚基因組為二倍化四倍體的特點。首先,本研究利用TALEN方法成功的對鯉魚sp7、runx2、spp1和mstn中的靶序列誘導了突變,并且有較高的突變效率;然后利用CRISPR-Cas9技術分別成功地修飾了鯉魚的兩個sp7基因,sp7a和sp7b,結果發(fā)現兩個基因的突變均造成了嚴重的骨骼缺陷。Micro-CT結果表明鯉魚sp7a和sp7b突變體的顱面骨和椎體骨發(fā)育均顯著慢于對照組,但是sp7a-CRISPR突變鯉魚比sp7b-CRISPR的突變體有更嚴重的骨骼發(fā)育缺陷。茜素紅染色結果表明sp7a-CRISPR突變體相對于對照組,表現出明顯的骨骼缺陷,包括鰓蓋和上頜骨的發(fā)育不全、血管脊椎不規(guī)則、椎體畸形以及肌間刺的長度較短等。而mstnba突變的鯉魚則在肌肉細胞的數量和肌肉纖維面積上相對于對照組出現了顯著增加,由此導致體重和體長顯著高于對照組。統(tǒng)計分析顯示mstnba-CRISPR組內突變體的體重和突變率呈明顯的正相關性。Western印跡表明CRISPR-Cas9介導的突變破壞了鯉魚骨骼肌的Mstn信號通路。qRT-PCR與HE染色結果共同表明mstnba-CRISPR突變的鯉魚F0代的肌肉纖維既表現出增生,也表現出肥大。我們還利用CRISPR-Cas9技術成功的構建了sp7a;mstnba鯉魚雙突變體,并且兩個基因的突變率均很高。這些研究表明了TALEN和CRISPR-Cas9技術均能夠高效的修飾鯉魚的基因組,為鯉魚遺傳研究和育種提供了新的途徑。另外,ENU誘變是一種可以改良農業(yè)物種遺傳性狀的有用方法。我們使用ENU處理鯉魚的成熟精子,然后體外受精得到ENU處理的F1代鯉魚。結果表明高濃度的ENU造成鯉魚胚胎畸形較多。使用合適濃度的ENU處理得到的F1代幼魚及成魚的體重分布范圍較大,并且其中大約16%的成年鯉魚表現出明顯的骨骼缺陷。這些結果表明ENU處理成熟精子也是一種改良鯉魚遺傳育種的有效方法。
[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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q953

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