Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
|Place of Conferral||北京|
|Keyword||脊尾白虾 显微注射 基因编辑 Crispr/cas9 几丁质酶 铁蛋白|
|Other Abstract|| 本文以脊尾白虾为研究对象，构建了脊尾白虾受精卵显微注射平台，成功实现了体外转录mRNA在脊尾白虾胚胎中表达。在脊尾白虾受精卵显微注射和外源mRNA在胚胎中成功表达的基础上，利用CRISPR/Cas9对脊尾白虾几丁质酶基因EcChi4进行了基因敲除，并初步检测了EcChi4敲除对脊尾白虾生长发育的影响。此外，结合本课题组已有的脊尾白虾转录组数据，利用RACE方法获得了脊尾白虾铁蛋白基因ferritin（EcFer）的全长cDNA序列；分析了EcFer在脊尾白虾胚胎不同发育时期和不同组织中的表达模式；通过金属离子刺激验证了EcFer对金属离子胁迫的应答；利用毕赤酵母表达系统对EcFer进行了体外重组表达，获得了重组蛋白，并进行了初步的活性分析。主要结果如下：|
1. The microinjection system for the embryos of E. carinicauda was successfully constructed. Using this system, the exogenous EGFP mRNA was injected into the single-cell embryos of E. carinicauda. The EGFP fluorescence was monitored 20 hour after injection. EGFP-mRNA-injected embryos displayed ubiquitous EGFP expression under the exciting light.
2. Based on the sequence of EcChi4, the gRNA target site was designed by the online tool ZiFiT. According to the gRNA target site, the DNA template of the gRNA was synthesized, and the gRNA was in vitro transcripted using the DNA template. On the other hand, the Cas9 mRNA was synthesized using the linearized plasmid pCMV-Cas9 as template. Using the microinjection system, Cas9 mRNA and gRNA were coinjected into the single-cell embryos of E. carinicauda. When the manipulated embryos developed into post larvae, the genome of the larvae was extracted, then the T7EI assay and Sanger sequencing were used to detect the mutation in the target loci of EcChi4. As the results shown, 50% of manipulated larvae have indels mutations on the target site of EcChi4. The statistic results of the weight, length and morphology of the mutant shrimps showed that, the mutations on EcChi4 did not have significant influence on the growth and development of E. carinicauda.
3. Based on the data of transcriptome and RACE technology, the full-length cDNA of ferritin from E. carinicauda (named EcFer) was obtained. An iron-responsive element (IRE) was identified on the 5’-UTR of EcFer. Using real-time PCR, the expression pattern of EcFer in different developmental stages and different tissues of shrimp were analyzed. In addition, the expression of EcFer was significantly up-regulated by metal ion stimulation. At last, EcFer was recombinantly expressed in Pichia pastoris and the activity of purified recombinant EcFer was identified.
|桂天书. 脊尾白虾CRISPR/Cas9基因编辑系统的建立及铁蛋白的功能分析[D]. 北京. 中国科学院大学,2016.|
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