脊尾白虾CRISPR/Cas9基因编辑系统的建立及铁蛋白的功能分析

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	脊尾白虾CRISPR/Cas9基因编辑系统的建立及铁蛋白的功能分析
	桂天书
学位类型	硕士
导师	张继泉
	2016-05-21
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	生物工程
关键词	脊尾白虾显微注射基因编辑 Crispr/cas9 几丁质酶铁蛋白
其他摘要	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：成功构建了脊尾白虾受精卵显微注射平台；并利用显微注射将体外转录的EGFP mRNA导入脊尾白虾单细胞受精卵中，受精卵发育20 h后在荧光显微镜下检测到外源EGFP mRNA在脊尾白虾受精卵中表达，在激发光激发下产生绿色荧光。根据EcChi4序列信息，利用在线工具ZiFiT设计对应gRNA靶点，合成对应gRNA的DNA模板，通过体外转录合成gRNA；以线性化的pCMV-Cas9质粒为模板，体外转录合成Cas9 mRNA。利用显微注射平台，将体外转录合成的Cas9 mRNA与gRNA共注射入脊尾白虾单细胞受精卵中。当胚胎发育至仔虾后，提取仔虾基因组，分别利用T7EI和Sanger sequencing对基因组进行检测，结果约50%的仔虾在EcChi4对应靶向位点发生了indels突变。统计虾的体重，体长，结合形态特征变化发现，EcChi4的indels突变未对脊尾白虾的生长发育产生明显的影响。利用脊尾白虾转录组数据信息结合RACE技术，克隆到脊尾白虾铁蛋白基因ferritin（EcFer）全长cDNA序列。分析EcFer的结构特征，发现位于其5’-UTR的铁离子应激元件（iron-responsive element，IRE）。利用real-time PCR方法确定了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。金属离子（Cd²⁺和Cu²⁺）刺激能使脊尾白虾EcFer的表达明显上调。利用毕赤酵母对EcFer进行了体外重组表达，并分离纯化得到了重组蛋白rEcFer，初步验证了其金属离子结合活性。 ; In this study, the microinjection system for the ridgetail white prawn Exopalaemon carinicauda was developed. Through this system, the in vitro synthesized RNAs were injected into the eggs of E. carinicauda and expressed in the embryos. Then on this basis, CRISPR/Cas9 system was used to knock out one chitinase gene of E. carinicauda (EcChi4). After the knocking-out of the EcChi4, the growth and development of E. carinicauda were monitored. On the other hand, based on the transcriptome data of E. carinicauda and RACE technology the full length cDNA of ferritin gene of E. carinicauda (EcFer) was cloned. Besides, the expression pattern in different developmental stages and different tissues of EcFer were analyzed. And the metal ion stress response of EcFer was also studied by metal ion stimulation. At last, EcFer was recombinantly expressed in Pichia pastoris and the activity of recombinant EcFer was identified. The main results are introduced as follows: 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.
学科领域	基因工程(亦称遗传工程)
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/112535
专题	实验海洋生物学重点实验室
推荐引用方式 GB/T 7714	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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