| 基于CRISPR/Cas9系统分析草地贪夜蛾Wnt1基因的生物学功能 |
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| 引用本文：付晓政,李瑞,邱琪琪,王梦珂,赵特,周琳.基于CRISPR/Cas9系统分析草地贪夜蛾Wnt1基因的生物学功能.植物保护学报,2023,50(1):59-70 |
| DOI：10.13802/j.cnki.zwbhxb.2023.2021059 |
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| 作者 | 单位 | E-mail | | 付晓政 | 河南农业大学植物保护学院, 河南省新型农药创制与应用重点实验室, 河南省绿色农药创制工程技术研究中心, 郑州 450002 | | | 李瑞 | 河南农业大学植物保护学院, 河南省新型农药创制与应用重点实验室, 河南省绿色农药创制工程技术研究中心, 郑州 450002 | | | 邱琪琪 | 河南农业大学植物保护学院, 河南省新型农药创制与应用重点实验室, 河南省绿色农药创制工程技术研究中心, 郑州 450002 | | | 王梦珂 | 河南农业大学植物保护学院, 河南省新型农药创制与应用重点实验室, 河南省绿色农药创制工程技术研究中心, 郑州 450002 | | | 赵特 | 河南农业大学植物保护学院, 河南省新型农药创制与应用重点实验室, 河南省绿色农药创制工程技术研究中心, 郑州 450002 | tezhao@126.com | | 周琳 | 河南农业大学植物保护学院, 河南省新型农药创制与应用重点实验室, 河南省绿色农药创制工程技术研究中心, 郑州 450002 | zhoulinhenau@163.com |
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| 中文摘要:为明确草地贪夜蛾Spodoptera frugiperda中Wnt1基因的生物学功能,采用实时荧光定量PCR(real-time quantitative PCR,qPCR)技术检测草地贪夜蛾胚胎发育期Wnt1基因的表达谱,利用CRISPR/Cas9系统对Wnt1基因进行定向敲除,并通过qPCR技术检测草地贪夜蛾Wnt1基因缺失突变体中与昆虫发育相关基因的表达水平。结果显示,Wnt1基因在草地贪夜蛾整个胚胎发育期均有表达,在胚胎发育早期(6h)表达水平最高。将SfWnt1-sgRNA和Cas9蛋白混合注入草地贪夜蛾卵中,胚胎死亡率明显升高,同时获得了Wnt1基因缺失的草地贪夜蛾突变体。突变率与注射浓度相关,注射浓度越高,突变率越高;当SfWnt1-sgRNA和Cas9蛋白混合后终浓度均为300 ng/μL时,注射的724粒卵中获得体节缺陷突变体52头,附肢缺陷突变体64头,组织特化突变体20头;SfWnt1-sg RNA和Cas9蛋白混合后终浓度均为100ng/μL时,注射的413粒卵中最终获得体节缺陷突变体14头,附肢缺陷突变体22头,组织特化突变体7头;这些突变体均无法存活至化蛹。草地贪夜蛾Wnt1基因被敲除后,与昆虫发育密切相关的17个基因表达水平均显著下调。表明Wnt1基因在草地贪夜蛾胚胎发育过程中发挥着至关重要的作用。 |
| 中文关键词:Wnt1基因 草地贪夜蛾 CRISPR/Cas9系统 胚胎发育 |
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| Biological function of fall armyworm Spodoptera frugiperda Wnt1 gene based on the CRISPR/Cas9 system |
| Author Name | Affiliation | E-mail | | FU Xiao-zheng | Henan Provincial Research Center for Green Pesticide Creation Engineering Technology, Henan Provincial Key Laboratory of New Pesticide Creation and Application, Department of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China | | | LI Rui | Henan Provincial Research Center for Green Pesticide Creation Engineering Technology, Henan Provincial Key Laboratory of New Pesticide Creation and Application, Department of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China | | | QIU Qi-qi | Henan Provincial Research Center for Green Pesticide Creation Engineering Technology, Henan Provincial Key Laboratory of New Pesticide Creation and Application, Department of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China | | | WANG Meng-ke | Henan Provincial Research Center for Green Pesticide Creation Engineering Technology, Henan Provincial Key Laboratory of New Pesticide Creation and Application, Department of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China | | | ZHAO Te | Henan Provincial Research Center for Green Pesticide Creation Engineering Technology, Henan Provincial Key Laboratory of New Pesticide Creation and Application, Department of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China | tezhao@126.com | | ZHOU Lin | Henan Provincial Research Center for Green Pesticide Creation Engineering Technology, Henan Provincial Key Laboratory of New Pesticide Creation and Application, Department of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China | zhoulinhenau@163.com |
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| Abstract:In order to clarify the biological function of Wnt1 gene in fall armyworm Spodoptera frugiperda, real-time quantitative PCR(qPCR) was used to detect the expression profile of Wnt1 gene during embryonic development of S. frugiperda, and the CRISPR/Cas9 system was used to knock out the Wnt1 gene of S. frugiperda and the expression levels of the genes related to insect development in S. frugiperda Wnt1 mutants were detected by qPCR. The results showed that Wnt1 was expressed in the whole embryonic developmental period of S. frugiperda, with the highest expression level in the early embryonic developmental stage(6 h). When SfWnt1-sgRNA and Cas9 protein were injected into S. frugiperda eggs, the embryonic lethality was significantly increased, and the Wnt1 gene-deficient mutants of S. frugiperda were obtained. The mutation rate was related to the injection concentration: the higher the injection concentration, the higher the mutation efficiency. When SfWnt1-sgRNA and Cas9 protein were mixed at a concentration of 300 ng/μL and injected into 724 eggs, 52 somite-defective mutants, 64 appendage-deficient mutants, and 20 tissue-specific mutants were obtained. When SfWnt1-sgRNA and Cas9 protein were mixed at a concentration of 100 ng/μL and injected into 413 eggs, 14 somite-defective mutants, 22 appendage-deficient mutants, and seven tissue-specific mutants were obtained. None of these mutants survived to pupation. After the S. frugiperda Wnt1 gene was knocked out, the expression levels of 17 genes closely related to insect development were significantly down-regulated. It indicated that Wnt1 gene played a crucial role in the embryonic development of S. frugiperda. |
| keywords:Wnt1 gene Spodoptera frugiperda CRISPR/Cas9 system embryonic development |
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