CRISPR/Cas9-Mediated Mutagenesis of <i>Antennapedia</i> in <i>Spodoptera frugiperda</i>
The homeotic gene <i>Antennapedia</i> (<i>Antp</i>) has been identified as playing a pivotal role in the morphogenesis of the thorax and wings across various insect species. Leveraging insights from previous studies, the functional characterization of <i>Antp</i>...
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MDPI AG
2023-12-01
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| Series: | Insects |
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| Online Access: | https://www.mdpi.com/2075-4450/15/1/16 |
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| author | Congke Wang Te Zhao Xiaolong Liu Tianliang Li Leiming He Qinqin Wang Li Wang Lin Zhou |
| author_facet | Congke Wang Te Zhao Xiaolong Liu Tianliang Li Leiming He Qinqin Wang Li Wang Lin Zhou |
| author_sort | Congke Wang |
| collection | DOAJ |
| description | The homeotic gene <i>Antennapedia</i> (<i>Antp</i>) has been identified as playing a pivotal role in the morphogenesis of the thorax and wings across various insect species. Leveraging insights from previous studies, the functional characterization of <i>Antp</i> in <i>S. frugiperda</i> was undertaken using RT-qPCR and the CRISPR/Cas9 genome-editing system. Phylogenetic analyses indicate that <i>Antp</i> shares a high degree of sequence homology among Lepidoptera species. The expression profile of <i>SfAntp</i> was detected by RT-qPCR. The results showed that <i>SfAntp</i> was expressed in the whole growth cycle of <i>S. frugiperda</i>, the expression level was the highest in the egg stage, and the expression level was higher from 12 h to 48 h. Tissue-specific expression profiling demonstrated that <i>SfAntp</i> was most abundantly expressed in the thoracic segments and legs. To functionally disrupt <i>SfAntp</i>, two sgRNA sites were designed at the first exon of <i>SfAntp</i> and the gene was knocked out by CRISPR/Cas9 via microinjection. The results showed that the deletion of <i>SfAntp</i> produced a mutant phenotype of thoracic fusion, thoracic leg defect, leg-like protrusions between the head and thoracic segments and pupation deformity. In addition, deletion of <i>SfAntp</i> resulted in high embryo mortality. Through DNA sequencing, it was found that the target site of the <i>SfAntp</i> mutant had different degrees of frameshift mutations, indicating that the mutant phenotype was indeed caused by the knockout of <i>SfAntp</i>. |
| first_indexed | 2024-03-08T10:47:06Z |
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| institution | Directory Open Access Journal |
| issn | 2075-4450 |
| language | English |
| last_indexed | 2024-03-08T10:47:06Z |
| publishDate | 2023-12-01 |
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| record_format | Article |
| series | Insects |
| spelling | doaj.art-47148bd5a26a451b94e1ae7e39c469742024-01-26T17:04:08ZengMDPI AGInsects2075-44502023-12-011511610.3390/insects15010016CRISPR/Cas9-Mediated Mutagenesis of <i>Antennapedia</i> in <i>Spodoptera frugiperda</i>Congke Wang0Te Zhao1Xiaolong Liu2Tianliang Li3Leiming He4Qinqin Wang5Li Wang6Lin Zhou7College of Plant Protection, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Plant Protection, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Plant Protection, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Plant Protection, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Plant Protection, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Plant Protection, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Plant Protection, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Plant Protection, Henan Agricultural University, Zhengzhou 450046, ChinaThe homeotic gene <i>Antennapedia</i> (<i>Antp</i>) has been identified as playing a pivotal role in the morphogenesis of the thorax and wings across various insect species. Leveraging insights from previous studies, the functional characterization of <i>Antp</i> in <i>S. frugiperda</i> was undertaken using RT-qPCR and the CRISPR/Cas9 genome-editing system. Phylogenetic analyses indicate that <i>Antp</i> shares a high degree of sequence homology among Lepidoptera species. The expression profile of <i>SfAntp</i> was detected by RT-qPCR. The results showed that <i>SfAntp</i> was expressed in the whole growth cycle of <i>S. frugiperda</i>, the expression level was the highest in the egg stage, and the expression level was higher from 12 h to 48 h. Tissue-specific expression profiling demonstrated that <i>SfAntp</i> was most abundantly expressed in the thoracic segments and legs. To functionally disrupt <i>SfAntp</i>, two sgRNA sites were designed at the first exon of <i>SfAntp</i> and the gene was knocked out by CRISPR/Cas9 via microinjection. The results showed that the deletion of <i>SfAntp</i> produced a mutant phenotype of thoracic fusion, thoracic leg defect, leg-like protrusions between the head and thoracic segments and pupation deformity. In addition, deletion of <i>SfAntp</i> resulted in high embryo mortality. Through DNA sequencing, it was found that the target site of the <i>SfAntp</i> mutant had different degrees of frameshift mutations, indicating that the mutant phenotype was indeed caused by the knockout of <i>SfAntp</i>.https://www.mdpi.com/2075-4450/15/1/16<i>Spodoptera frugiperda</i><i>Antp</i>CRISPR/Cas9thoracic segment developmentthoracic leg |
| spellingShingle | Congke Wang Te Zhao Xiaolong Liu Tianliang Li Leiming He Qinqin Wang Li Wang Lin Zhou CRISPR/Cas9-Mediated Mutagenesis of <i>Antennapedia</i> in <i>Spodoptera frugiperda</i> Insects <i>Spodoptera frugiperda</i> <i>Antp</i> CRISPR/Cas9 thoracic segment development thoracic leg |
| title | CRISPR/Cas9-Mediated Mutagenesis of <i>Antennapedia</i> in <i>Spodoptera frugiperda</i> |
| title_full | CRISPR/Cas9-Mediated Mutagenesis of <i>Antennapedia</i> in <i>Spodoptera frugiperda</i> |
| title_fullStr | CRISPR/Cas9-Mediated Mutagenesis of <i>Antennapedia</i> in <i>Spodoptera frugiperda</i> |
| title_full_unstemmed | CRISPR/Cas9-Mediated Mutagenesis of <i>Antennapedia</i> in <i>Spodoptera frugiperda</i> |
| title_short | CRISPR/Cas9-Mediated Mutagenesis of <i>Antennapedia</i> in <i>Spodoptera frugiperda</i> |
| title_sort | crispr cas9 mediated mutagenesis of i antennapedia i in i spodoptera frugiperda i |
| topic | <i>Spodoptera frugiperda</i> <i>Antp</i> CRISPR/Cas9 thoracic segment development thoracic leg |
| url | https://www.mdpi.com/2075-4450/15/1/16 |
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