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| Obtaining and Photosynthetic Characteristics Analysis of SmCPS1 and SmCYP76AH1 Double-gene Overexpressed Transgenic Lines of Salvia miltiorrhiza |
| GUO Guang-Yang, LI Yi-Long, XU Hua-Wei, WANG Ting, HOU Dian-Yun*, LYU Shu-Fang* |
| College of Agriculture, Henan University of Science and Technology, Luoyang 471000, China |
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Abstract Salvia miltiorrhiza is a traditional Chinese herbal medicine, and its bioactive ingredients mainly include tanshinones and salvianolic acids. In order to explore the effects of multi-gene co-overexpression on the growth and accumulation of active substances in S. miltiorrhiza, in this study, the tissue expression and protein subcellular localization of cobacyl pyrophosphate synthetase gene 1 (SmCPS1) and cytochrome P450 monooxygenase gene (SmCYP76AH1) were analyzed, and pYLTAC380H-SmCPS1-SmCYP76AH1, an co-overexpression vector of SmCPS1 and SmCYP76AH1, was constructed using a multi-gene stacking expression system, and then the overexpression vector was transformed to S. miltiorrhiza clean seedlings by Agrobacterium tumefaciens-mediated method. Through callus induction culture, resistance and green fluorescence signal screening, genome integration identification and gene transcription level detection after transformation, double-gene co-overexpressed S. miltiorrhiza lines were obtained, and the content of tanshinone, photosynthetic and fluorescence characteristics of the double-gene co-overexpression lines were further determined. qRT-PCR showed that SmCPS1 and SmCYP76AH1 were expressed in the roots, stems and leaves of S. miltiorrhiza, both of which were highly expressed in the roots. Subcellular localization analysis showed that SmCPS1 was localized in chloroplasts and cytoplasm, and SmCYP76AH1 was localized in endoplasmic reticulum. The contents of tanshinone Ⅰ, tanshinone ⅡA and cryptotanshinone in roots of SmCPS1 and SmCYP76AH1 double-gene co-overexpression lines were extremely significantly increased (P≤0.01). Photosynthetic and fluorescence characteristics of leaves showed that the transpiration rate and stomatal conductance decreased significantly in SmCPS1 and SmCYP76AH1 co-overexpression lines, and the intercellular CO2 concentration (Ci) and net photosynthetic rate (Pn) varied with the expression level of SmCPS1 and SmCYP76AH1. The initial fluorescence (F0), maximum fluorescence (Fm) and non-photochemical quenching coefficient (NPQ) increased significantly in co-overexpressed lines, but the maximum photochemical efficiency (Fv/Fm) of photosystem Ⅱ (PSⅡ) remained unchanged. The multigene transformation method was established in this study and the double-gene co-overexpression lines were obtained which provides experimental materials and methods for further research on the regulation of tanshinone, and provide reference for exploring the function of of SmCPS1 and SmCYP76AH1 in the above ground part of S. miltiorrhiza.
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Received: 14 December 2023
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Corresponding Authors:
* dianyun518@163.com; lvsf780515@haust.edu.cn
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