CRISPR/Cas9技术对脊尾白虾蜕皮抑制激素基因的靶向敲除研究

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	CRISPR/Cas9技术对脊尾白虾蜕皮抑制激素基因的靶向敲除研究
	宋凤阁
学位类型	硕士
导师	张继泉
	2017-05-18
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 Crispr/cas9 蜕皮抑制激素基因编辑
其他摘要	甲壳动物的蜕皮与生长密切相关。已有的研究表明蜕皮抑制激素MIH在甲壳动物蜕皮过程中起着负调节作用。但是，由于甲壳动物功能基因研究平台的限制，迄今为止未见在活体内通过基因敲除的方法对甲壳动物MIH基因功能进行研究的报道。在前期研究中，本课题组成功实现了基于CRISPR/Cas9技术的脊尾白虾基因组编辑。本论文以脊尾白虾为研究对象，在其转录组数据基础上，成功获得了脊尾白虾MIH（EcMIH）的全长序列，并对该基因表达特征进行了分析。在此基础上，通过共注射体外转录的Cas9 mRNA和gRNA的方法成功敲除了EcMIH，获得幼体发育时间缩短的脊尾白虾个体，同时对其功能进行了初步研究。此外，本研究成功构建了Cas9核酸内切酶的分泌型表达载体pCT7-CHISP6H-Cas9，制备了Cas9重组蛋白，并在体外检测了其对脊尾白虾基因组编辑的可行性。主要结果归纳为以下两个方面： 1. 通过对脊尾白虾转录组数据分析和PCR验证确定了脊尾白虾EcMIH的基因信息，获得了该基因的全长序列。EcMIH的开放阅读框编码119个氨基酸，预测的分子量为13747.82 Da，等电点8.61，其基因由3个外显子和2个内含子组成。荧光实时定量PCR检测了EcMIH在不同组织、不同发育时期以及不同蜕皮时期的表达特征。另外，在第二个外显子上设计并体外转录合成EcMIH gRNA，通过脊尾白虾胚胎显微注射的方法将体外转录的Cas9 mRNA和EcMIH gRNA共注射脊尾白虾单细胞时期受精卵。在EcMIH gRNA靶标位点两侧设计检测引物对扩增目的片段，PCR产物测序以及单克隆检测分析结果均表明实验组脊尾白虾个体的基因靶标位点发生了碱基缺失突变，突变率达42.9%。对EcMIH功能初步分析结果显示，基因被敲除后的脊尾白虾幼体发育至仔虾的时间由11天减少为8天，而其形态与生长发育等与野生型脊尾白虾相比没有明显差异。 2. 以Cas9P-1EA为模板设计引物扩增Cas9成熟肽序列，根据In-fusion^RHD Clontech kit将其和PmaC I酶切的pCT7-CHISP6H线性化质粒进行反应构建分泌型表达载体pCT7-CHISP6H-Cas9。将重组质粒转化至大肠杆菌BL21（DE3）进行重组表达，Western blot检测分析获得分泌表达的Cas9核酸内切酶。亲和层析以及脱盐纯化得到具有生物活性的重组蛋白，并且该重组蛋白可以在gRNA的指导下体外切割靶标质粒，分析了其对脊尾白虾基因组编辑的可行性。; Molting is a crucial process for crustaceans which has a close relationship with growth. Early researches showed that molt-inhibiting hormone (MIH) was a negative-regulatory factor that suppresses its molting process in the complicated crustacean molting pathway. But because of the limited crustacean functional gene research platform, no studies on crustacean MIH gene in vivo have been reported so far. We have successfully applied the CRISPR/Cas9 technology on the genome editing of the ridgetail white prawn Exopalaemon carinicauda in our previous studies. In this study, transcriptome of E. carinicauda were analyzed and the molt-inhibiting hormone gene (EcMIH) information was confirmed by PCR. The expression profile of EcMIH was detected by qPCR, and it was successfully knocked out through the genome editing tool CRISPR/Cas9. The obtained mutations had a three days shorter time before developing into juveniles compared with blank and control groups, which verified the EcMIH function preliminarily. In addition, the secretory expression vector pCT7-CHISP6H-Cas9 was constructed and the recombinant Cas9 protein was prepared. Moreover, the biological activity of recombinant Cas9 in genome editing was studied in vitro at the same time. 1. Based on the transcriptome of E.carinicauda, the full-length cDNA sequence of EcMIH was obtained. The ORF of it encoded 119 amino acids with a predicted molecular weight (MW) about 13747.82 Da. The theoretical isoelectric point (PI) of EcMIH was 8.61. It consists of three exons and two introns. The expression profiles of EcMIH were analyzed by real-time PCR in different tissues, different stages and different molting periods. EcMIH gRNA was designed in the second exon and was transcript synthesized in vitro, then it was injected into E.carinicauda one-cell embryos combined with mRNA of Cas9 nuclease. The detection primers were designed and the genomic region flanking the EcMIH gRNA target was amplified. Both the sequencing results of PCR products and monoclonal assay analysis showed that EcMIH successfully knocked out by using CRISPR/Cas9 system. The frameshift mutant E. carinicauda individuals were obtained, and the mutation ratio reached 42.9%. In addition, the larvae development time of these mutants was reduced from 11 days to 8 days, while no other significant differences were observed. 2. The mature peptide sequence of Cas9 was amplified with designed pCT7-CHISP6H-Cas9F/R as primers and Cas9P-1EA as template. Then according to the In-fusion^RHD Clontech kit, the PCR products and the linearized pCT7-CHISP6H plasmids digested by PmaC I were put together to construct the recombinant secretory expression plasmid pCT7-CHISP6H-Cas9. And the secretory recombinant Cas9 was obtained after the pCT7-CHISP6H-Cas9 plasmid was transformed into BL21(DE3). The rCas9 protein was purified by affinity chromatography and desalting methods. Moreover, the recombinant enzyme could cleave the target plasmid under the guidance of gRNA in vitro.
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136613
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	宋凤阁. CRISPR/Cas9技术对脊尾白虾蜕皮抑制激素基因的靶向敲除研究[D]. 北京. 中国科学院大学,2017.

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