Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensis
Abstract Background Molting is an important physiological process in the growth and development of arthropoda, which is mainly regulated by juvenile hormone and ecdysone. CYP302A1 is a key enzyme which plays a critical role in the synthesis of ecdysone in insects, but it has not been identified in c...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-01-01
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| Series: | Frontiers in Zoology |
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| Online Access: | https://doi.org/10.1186/s12983-023-00483-2 |
| _version_ | 1797952445812310016 |
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| author | Huiying Qi Huijuan Cao Yajie Zhao Yaqin Cao Qide Jin Yeping Wang Kun Zhang Daogui Deng |
| author_facet | Huiying Qi Huijuan Cao Yajie Zhao Yaqin Cao Qide Jin Yeping Wang Kun Zhang Daogui Deng |
| author_sort | Huiying Qi |
| collection | DOAJ |
| description | Abstract Background Molting is an important physiological process in the growth and development of arthropoda, which is mainly regulated by juvenile hormone and ecdysone. CYP302A1 is a key enzyme which plays a critical role in the synthesis of ecdysone in insects, but it has not been identified in cladocera. Results The CYP302Al gene of Daphnia sinensis was cloned and its function was analyzed in this paper. The CYP302Al gene of D. sinensis was 5926 bp in full-length, with an open reading frame (ORF) of 1596 bp that encoded 531 amino acids (aa), a molecular weight of 60.82 kDa and an isoelectric point of 9.29. The amino acid sequence analysis revealed that there were five characteristic conserved regions of cytochrome P450 family (namely helix-C, helix-K, helix-I, PERF and heme-binding). In dsRNA mediated experiment, the expression level of CYP302A1 gene decreased significantly (knock-down of 56.22%) in the 5% Escherichia coli concentration treatment. In addition, the expression levels of EcR and USP and HR3 genes in the downstream were also significantly decreased, whereas that of FTZ-f1 gene increased significantly. In the 5% E. coli treatment, the molting time at maturity of D. sinensis prolonged, and the development of embryos in the incubation capsule appeared abnormal or disintegrated. The whole-mount in situ hybridization showed that the CYP302A1 gene of D. sinensis had six expression sites before RNA interference (RNAi), which located in the first antennal ganglion, ovary, cecae, olfactory hair, thoracic limb and tail spine. However, the expression signal of the CYP302A1 gene of D. sinensis disappeared in the first antennal ganglion and obviously attenuated in the ovary after RNAi. Conclusion The CYP302A1 gene played an important role in the ecdysone synthesis pathway of D. sinensis, and the knock-down of the gene affected the molting and reproduction of D. sinensis. |
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| format | Article |
| id | doaj.art-ce781ca37ab34ed7a1128674f9f0ae75 |
| institution | Directory Open Access Journal |
| issn | 1742-9994 |
| language | English |
| last_indexed | 2024-04-10T22:46:25Z |
| publishDate | 2023-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Frontiers in Zoology |
| spelling | doaj.art-ce781ca37ab34ed7a1128674f9f0ae752023-01-15T12:17:13ZengBMCFrontiers in Zoology1742-99942023-01-0120111110.1186/s12983-023-00483-2Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensisHuiying Qi0Huijuan Cao1Yajie Zhao2Yaqin Cao3Qide Jin4Yeping Wang5Kun Zhang6Daogui Deng7School of Life Science, Huaibei Normal UniversitySchool of Life Science, Huaibei Normal UniversitySchool of Life Science, Huaibei Normal UniversitySchool of Life Science, Huaibei Normal UniversitySchool of Life Science, Huaibei Normal UniversitySchool of Life Science, Huaibei Normal UniversitySchool of Life Science, Huaibei Normal UniversitySchool of Life Science, Huaibei Normal UniversityAbstract Background Molting is an important physiological process in the growth and development of arthropoda, which is mainly regulated by juvenile hormone and ecdysone. CYP302A1 is a key enzyme which plays a critical role in the synthesis of ecdysone in insects, but it has not been identified in cladocera. Results The CYP302Al gene of Daphnia sinensis was cloned and its function was analyzed in this paper. The CYP302Al gene of D. sinensis was 5926 bp in full-length, with an open reading frame (ORF) of 1596 bp that encoded 531 amino acids (aa), a molecular weight of 60.82 kDa and an isoelectric point of 9.29. The amino acid sequence analysis revealed that there were five characteristic conserved regions of cytochrome P450 family (namely helix-C, helix-K, helix-I, PERF and heme-binding). In dsRNA mediated experiment, the expression level of CYP302A1 gene decreased significantly (knock-down of 56.22%) in the 5% Escherichia coli concentration treatment. In addition, the expression levels of EcR and USP and HR3 genes in the downstream were also significantly decreased, whereas that of FTZ-f1 gene increased significantly. In the 5% E. coli treatment, the molting time at maturity of D. sinensis prolonged, and the development of embryos in the incubation capsule appeared abnormal or disintegrated. The whole-mount in situ hybridization showed that the CYP302A1 gene of D. sinensis had six expression sites before RNA interference (RNAi), which located in the first antennal ganglion, ovary, cecae, olfactory hair, thoracic limb and tail spine. However, the expression signal of the CYP302A1 gene of D. sinensis disappeared in the first antennal ganglion and obviously attenuated in the ovary after RNAi. Conclusion The CYP302A1 gene played an important role in the ecdysone synthesis pathway of D. sinensis, and the knock-down of the gene affected the molting and reproduction of D. sinensis.https://doi.org/10.1186/s12983-023-00483-2D. sinensisEcdysoneCYP302A1 geneCloningFunctional analysis |
| spellingShingle | Huiying Qi Huijuan Cao Yajie Zhao Yaqin Cao Qide Jin Yeping Wang Kun Zhang Daogui Deng Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensis Frontiers in Zoology D. sinensis Ecdysone CYP302A1 gene Cloning Functional analysis |
| title | Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensis |
| title_full | Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensis |
| title_fullStr | Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensis |
| title_full_unstemmed | Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensis |
| title_short | Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensis |
| title_sort | cloning and functional analysis of the molting gene cyp302a1 of daphnia sinensis |
| topic | D. sinensis Ecdysone CYP302A1 gene Cloning Functional analysis |
| url | https://doi.org/10.1186/s12983-023-00483-2 |
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