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| OfSPL Genes Cloning and Its Expression Analysis During Flower Bud Differentiation at Different Temperature in Osmanthus fragrans |
| WU Hong-Fei1,2,3, ZHOU Min-Shu1,2,3, ZHU Shou-Kuo1,2,3, YANG Li-Yuan1,2,3, ZHAO Hong-Bo1,2,3,*, DONG Bin1,2,3,* |
1 School of Landscape Architecture, Zhejiang A&F University, Hangzhou 311300, China;
2 Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants, Zhejiang Agriculture & Forestry University, Hangzhou 311300, China;
3 Key Laboratory of National Forestry and Grassland Administration on Germplasm Innovation and Utilization for Southern Garden Plants, Zhejiang Agriculture & Forestry University, Hangzhou 311300, China |
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Abstract Ambient temperature is significantly affected flower bud development and flower opening of sweet osmanthus (Osmanthus fragrans). To further understand SQUAMOSA promoter-binding protein-like (SPL) genes and miR156 (microRNA156) on how to respond to ambient temperature in regulating flower bud development of sweet osmanthus, the variety of Osmanthus fragrans 'Yanhonggui' were selected, and 10 OfSPLs (OfSPL1A: MT756841; OfSPL1B: MT756842; OfSPL5: MT756843; OfSPL6: MT756844; OfSPL7: MT756845; OfSPL8: MT756849; OfSPL10: MT756846; OfSPL11: MT756847; OfSPL12: MT756848; OfSPL13: MT756850) were cloned by PCR. Meanwhile, the expression of miR156 and OfSPLs were identified by real-time quantitative PCR (qRT-PCR) to further analyze their temporal and spatial expression patterns. The results of OfSPL sequences analysis revealed that the cloned OfSPLs cDNA length between 1 004~3 824 bp, the sizes of OfSPL proteins ranged from 232 amino acid (aa) to 1 006 aa, and all of OfSPLs included a conserved SBP domain. Furthermore, all of OfSPL genes showed high homology with OlSPL genes from the species of Olea europaea by amino acid sequence alignment and evolutionary analysis. To investigate the intracellular localization of the proteins coded by the OfSPL genes, the bioinformatics method was used for predicting. The predicted results showed that all of the OfSPL proteins located in the nucleus. miR156 plays its role through regulating SPL gene expression by translational repression or degradation of mRNA in a sequence-specific manner. Therefore, the binding site of SPL genes was further analyzed. The expression of OfPL6, OfPL10, OfPL11, and OfSPL12 might be affected by miR156 through targeting their binding sites. Under different temperature treatments, the expression of OfSPL1A, OfSPL5, OfSPL6, OfSPL10, and OfSPL13 significantly increased at 19 ℃ comparing with the control 25 ℃. In contrast, the expression of miR156-1 and miR156-2 under 19 ℃ treatment were lower than the control 25 ℃. The results indicated that the expression of miR156-1 and miR156-2 may be inhibited by low ambient temperature, and resulted in the genes of OfSPL6 and OfSPL10 upregulation and then promoted the flower bud differentiation. Additionally, OfSPL1A, OfSPL5, and OfSPL13 may also involve in the flower bud development of O. fragrans at low ambient temperature, but they were not directly regulated by miR156-1 and miR156-2. This study could provide a reference for further research on the regulation of flower bud differentiation and flowering time by ambient temperature in O. fragrans.
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Received: 03 February 2020
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Corresponding Authors:
*zhaohb@zafu.edu.cn;dongbin@zafu.edu.cn
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