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Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius
OBJECTIVE: Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10230621/ https://www.ncbi.nlm.nih.gov/pubmed/37265765 http://dx.doi.org/10.1016/j.chmed.2022.12.005 |
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author | Tang, Xiaohui Ren, Chaoxiang Hu, Jing Chen, Jiang Wang, Jie Wang, Rui Wu, Qinghua Liao, Wan Pei, Jin |
author_facet | Tang, Xiaohui Ren, Chaoxiang Hu, Jing Chen, Jiang Wang, Jie Wang, Rui Wu, Qinghua Liao, Wan Pei, Jin |
author_sort | Tang, Xiaohui |
collection | PubMed |
description | OBJECTIVE: Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs in the molecular characterization and enzyme activities were investigated. METHODS: Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C. tinctorius. Chalcone biosynthase genes in C. tinctorius (CtCHSs) were cloned from cDNA of flowers of C. tinctorius. The cloned gene sequences were analyzed by bioinformatics, and their expression patterns were analyzed by real-time PCR (RT-PCR). The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot. A recombinant vector of CtCHS was constructed. The CtCHS recombinant protein was induced and purified to detect the enzyme reaction (catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone). The reaction product was detected by HPLC and LC-MS. RESULTS: Two full-length CtCHS genes were successfully cloned from the flowers of safflower (CtCHS1 and CtCHS3), with gene lengths of 1525 bp and 1358 bp, respectively. RT-PCR analysis showed that both genes were highly expressed in the flowers, but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers. WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers. HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone. CONCLUSION: CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower. |
format | Online Article Text |
id | pubmed-10230621 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-102306212023-06-01 Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius Tang, Xiaohui Ren, Chaoxiang Hu, Jing Chen, Jiang Wang, Jie Wang, Rui Wu, Qinghua Liao, Wan Pei, Jin Chin Herb Med Original Article OBJECTIVE: Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs in the molecular characterization and enzyme activities were investigated. METHODS: Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C. tinctorius. Chalcone biosynthase genes in C. tinctorius (CtCHSs) were cloned from cDNA of flowers of C. tinctorius. The cloned gene sequences were analyzed by bioinformatics, and their expression patterns were analyzed by real-time PCR (RT-PCR). The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot. A recombinant vector of CtCHS was constructed. The CtCHS recombinant protein was induced and purified to detect the enzyme reaction (catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone). The reaction product was detected by HPLC and LC-MS. RESULTS: Two full-length CtCHS genes were successfully cloned from the flowers of safflower (CtCHS1 and CtCHS3), with gene lengths of 1525 bp and 1358 bp, respectively. RT-PCR analysis showed that both genes were highly expressed in the flowers, but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers. WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers. HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone. CONCLUSION: CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower. Elsevier 2023-02-16 /pmc/articles/PMC10230621/ /pubmed/37265765 http://dx.doi.org/10.1016/j.chmed.2022.12.005 Text en © 2022 Tianjin Press of Chinese Herbal Medicines. Published by ELSEVIER B.V. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Original Article Tang, Xiaohui Ren, Chaoxiang Hu, Jing Chen, Jiang Wang, Jie Wang, Rui Wu, Qinghua Liao, Wan Pei, Jin Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius |
title | Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius |
title_full | Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius |
title_fullStr | Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius |
title_full_unstemmed | Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius |
title_short | Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius |
title_sort | cloning, expression and activity analysises of chalcone synthase genes in carthamus tinctorius |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10230621/ https://www.ncbi.nlm.nih.gov/pubmed/37265765 http://dx.doi.org/10.1016/j.chmed.2022.12.005 |
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