Selection and validation of reference genes for quantitative real-time PCR during the developmental stages of seeds in Sophora davidii
IntroductionQuinolizidine alkaloids, such as matrine and sophocarpine, enriched in Sophora davidii seeds, demonstrate notable anticancer properties. However, the biosynthetic pathway of these alkaloids remains incompletely elucidated, and the expression patterns of key enzyme genes involved in this...
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Frontiers Media S.A.
2025-05-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2025.1485586/full |
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| author | Jingjing Li Jingjing Li Shanrong Han Shanrong Han Zongren Xu Bin Deng Na Zheng Na Zheng Yaqiong Su Yaqiong Su Ziyao Qiao Ziyao Qiao Yun Yang Yun Yang Hong Zhang Zongsuo Liang Jing Liu Shuai Liu |
| author_facet | Jingjing Li Jingjing Li Shanrong Han Shanrong Han Zongren Xu Bin Deng Na Zheng Na Zheng Yaqiong Su Yaqiong Su Ziyao Qiao Ziyao Qiao Yun Yang Yun Yang Hong Zhang Zongsuo Liang Jing Liu Shuai Liu |
| author_sort | Jingjing Li |
| collection | DOAJ |
| description | IntroductionQuinolizidine alkaloids, such as matrine and sophocarpine, enriched in Sophora davidii seeds, demonstrate notable anticancer properties. However, the biosynthetic pathway of these alkaloids remains incompletely elucidated, and the expression patterns of key enzyme genes involved in this pathway require further investigation. Quantitative real-time PCR (qRT-PCR) serves as a highly sensitive method for gene expression analysis, yet selecting appropriate reference genes is crucial to ensure the accuracy and reliability of results.MethodsTen candidate reference genes (18S, ACT13, RL15B, RL74, RLA2, RL182, RL291, EF1-α, EF1G, and YLS8) were evaluated for their expression stability in Sophora davidii seeds collected at five distinct developmental stages post-flowering, characterized by significant morphological changes. Five computational tools—GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder—were employed to comprehensively analyze the stability of these genes.ResultsAmong the candidate genes, EF1G and RL291 exhibited the highest expression stability, whereas RL182 proved unsuitable as a reference gene. Validation experiments confirmed that normalization using stable reference genes (e.g., EF1G and RL291) yielded accurate quantification of target gene expression.DiscussionThis study identifies EF1G and RL291 as optimal reference genes for qRT-PCR analysis during Sophora davidii seed development, addressing a critical methodological gap in alkaloid biosynthesis research. These findings underscore the necessity of rigorous reference gene validation to ensure reliable gene expression data. The results advance our understanding of quinolizidine alkaloid biosynthesis and highlight the broader importance of reference gene selection in plant molecular studies. |
| format | Article |
| id | doaj-art-cc926a46bb3a4abaa34b0fbeee442bd6 |
| institution | Kabale University |
| issn | 1664-462X |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj-art-cc926a46bb3a4abaa34b0fbeee442bd62025-08-20T03:49:46ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2025-05-011610.3389/fpls.2025.14855861485586Selection and validation of reference genes for quantitative real-time PCR during the developmental stages of seeds in Sophora davidiiJingjing Li0Jingjing Li1Shanrong Han2Shanrong Han3Zongren Xu4Bin Deng5Na Zheng6Na Zheng7Yaqiong Su8Yaqiong Su9Ziyao Qiao10Ziyao Qiao11Yun Yang12Yun Yang13Hong Zhang14Zongsuo Liang15Jing Liu16Shuai Liu17Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education/College of Life Science, Northwest University, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaKey Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education/College of Life Science, Northwest University, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaSchool of Pharmacy, Guizhou University of Traditional Chinese Medicine, Guiyang, ChinaKey Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education/College of Life Science, Northwest University, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaKey Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education/College of Life Science, Northwest University, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaKey Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education/College of Life Science, Northwest University, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaKey Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education/College of Life Science, Northwest University, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaInstitute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi’an, Shaanxi, ChinaIntroductionQuinolizidine alkaloids, such as matrine and sophocarpine, enriched in Sophora davidii seeds, demonstrate notable anticancer properties. However, the biosynthetic pathway of these alkaloids remains incompletely elucidated, and the expression patterns of key enzyme genes involved in this pathway require further investigation. Quantitative real-time PCR (qRT-PCR) serves as a highly sensitive method for gene expression analysis, yet selecting appropriate reference genes is crucial to ensure the accuracy and reliability of results.MethodsTen candidate reference genes (18S, ACT13, RL15B, RL74, RLA2, RL182, RL291, EF1-α, EF1G, and YLS8) were evaluated for their expression stability in Sophora davidii seeds collected at five distinct developmental stages post-flowering, characterized by significant morphological changes. Five computational tools—GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder—were employed to comprehensively analyze the stability of these genes.ResultsAmong the candidate genes, EF1G and RL291 exhibited the highest expression stability, whereas RL182 proved unsuitable as a reference gene. Validation experiments confirmed that normalization using stable reference genes (e.g., EF1G and RL291) yielded accurate quantification of target gene expression.DiscussionThis study identifies EF1G and RL291 as optimal reference genes for qRT-PCR analysis during Sophora davidii seed development, addressing a critical methodological gap in alkaloid biosynthesis research. These findings underscore the necessity of rigorous reference gene validation to ensure reliable gene expression data. The results advance our understanding of quinolizidine alkaloid biosynthesis and highlight the broader importance of reference gene selection in plant molecular studies.https://www.frontiersin.org/articles/10.3389/fpls.2025.1485586/fullSophora davidiireference genedevelopment stages of seedsstabilitynormalization |
| spellingShingle | Jingjing Li Jingjing Li Shanrong Han Shanrong Han Zongren Xu Bin Deng Na Zheng Na Zheng Yaqiong Su Yaqiong Su Ziyao Qiao Ziyao Qiao Yun Yang Yun Yang Hong Zhang Zongsuo Liang Jing Liu Shuai Liu Selection and validation of reference genes for quantitative real-time PCR during the developmental stages of seeds in Sophora davidii Frontiers in Plant Science Sophora davidii reference gene development stages of seeds stability normalization |
| title | Selection and validation of reference genes for quantitative real-time PCR during the developmental stages of seeds in Sophora davidii |
| title_full | Selection and validation of reference genes for quantitative real-time PCR during the developmental stages of seeds in Sophora davidii |
| title_fullStr | Selection and validation of reference genes for quantitative real-time PCR during the developmental stages of seeds in Sophora davidii |
| title_full_unstemmed | Selection and validation of reference genes for quantitative real-time PCR during the developmental stages of seeds in Sophora davidii |
| title_short | Selection and validation of reference genes for quantitative real-time PCR during the developmental stages of seeds in Sophora davidii |
| title_sort | selection and validation of reference genes for quantitative real time pcr during the developmental stages of seeds in sophora davidii |
| topic | Sophora davidii reference gene development stages of seeds stability normalization |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2025.1485586/full |
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