利用CRISPRCas9技术构建棉铃虫UGT41B3和UGT40D1基因敲除品系
毕业论文关键词：CRISPR/Cas9；棉铃虫；UDP-糖基转移酶；UGT41B3；UGT40D1
Construction of UGT41B3 and UGT40D1 knockout line in Helicoverpa armigera utilizing the CRISPR/Cas9 system
Abstract：Helicoverpa armigera has a wide host range, and its larvae have strong ability of detoxification, which could metabolize the defensive secondary metabolites from various plants. Among them, H.armigera could use UDP-Glycosyltransferase (UGT), which is related to the UGT41B3 and UGT40D1 gene products , to glycosylate the cotton secondary metabolite --- gossypol. CRISPR/Cas9 system is a powerful gene editing tool which was been discovered and developed recently, the system can lead to genomic DNA double strand breaks in specific gene loci, achieving the purpose of gene editing by biological non homologous end joining and homologous recombination repairing. In this study the UGT41B3 and UGT40D1 genes were knockouted using CRISPR/Cas9 and the UGT41B3 and UGT40D1 gene knockout lines were constructed, which can provide the basis to reveal the role of these two genes in detoxification of gossypol and insecticide in H.armigera.
Key words: CRISPR/Cas9；Helicoverpa armigera；UDP-Glycosyltransferase；UGT41B3；UGT40D1
目 录
摘要1
关键词1
Abstract1
Key words1
引言（或绪论）1
1材料与方法2
1.1实验材料 2
1.1.1供试昆虫2
1.1.2主要仪器和试剂2
1.2实验方法 2
1.2.1 sgRNA设计2
1.2.2胚胎期显微注射5
1.2.3 UGT41B3和UGT40D1基因敲除基因型检测5
1.2.4 构建UGT41B3基因敲除品系5
1.2.5 构建UGT40D1基因敲除品系6
2 结果与分析6
2.1 UGT41B3基因的基因编辑效率6
2.2 UGT41B3的基因编辑突变类型6
2.3 UGT41B3基因突变遗传到下一代的效率7
2.4 UGT41B3基因敲除品系的构建7
2.5 UGT40D3基因敲除品系的构建7
3讨论8
致谢9
参考文献10
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图26
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图48
表17
利用CRISPR/Cas9技术构建棉铃虫UGT41B3和UGT40D1基因敲除品系
棉铃虫Helicoverpa armigera（Hübner）属鳞翅目Lepidoptera，夜蛾科Noctuidae，棉铃虫属Helicoverpa Hardwick，广泛分布于南北纬40°之间的亚洲、非洲、欧洲、大洋洲和太平洋西南部岛屿[1-2]。棉铃虫的寄主范围广，据报道，在我国棉铃虫能够取食20多个科200余种植物，寄主多样性能够使棉铃虫适应于各种不稳定的环境，为种群暴发提供基础。
棉铃虫幼虫具有较强的解毒代谢能力,能够降解多种植物的防御性次生物质，在许多情况下其过程可分为三个阶段。第一阶段，细胞色素P450、羧酸酯酶等可以直接作用于这些次生物质分子，引入或释放功能基团从而提高其水溶性；第二阶段，UDP糖基转移酶（UDP-glycosyltransferase,UGT）、谷胱甘肽S-转移酶等能通过将內源分子结合到次生物质上，减少次生物质活性并增加其水溶性，从而使其失去在生物膜间扩散的能力[3]；在第三阶段，ATP结合转运蛋白可以帮助结合了内源分子的次生物质跨膜运输并使其排泄，以达到解毒的作用。
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