| Sweet osmanthus(Osmanthus fragrans Lour.),belonging to the family Oleaceae,is an evergreen tree or shrub as well as one of the top ten traditional famous flowers in China.(1)According to the different flower color,Osmanthus fragrans is divided into Yingui group,Jingui group,Dangui group and Sijigui group.The flower color of Osmanthus fragrans differs greatly among different cultivars,because the kinds of carotenoids and their contents are different among different cultivars.At present,carotenoid biosynthesis,as well as the related genes,have been well elucidated in many plants,but the regulation of the genes in the petals of Osmanthus fragrans is not clear.By HPLC and transcriptome sequencing analysis,the key genes and their candidate transcription factors(TFs)affecting the carotenoid metabolism pathway of Osmanthus fragrans were explored,and the regulatory network between the key genes and the candidate TFs was established,which provided useful information for the in-depth study of the potential transcriptional regulation mechanism of carotenoid accumulation in Osmanthus fragrans.(2)9-Cis-Epoxycarotenoid Dioxygenase 3(NCED3)is a key enzyme in the carotenoid metabolic pathway as well as a key enzyme in ABA biosynthesis in higher plants.Studies have shown that high temperature,drought and salt stress can induce the expression of At NCED3,lead to the increase of ABA content and finally improve the tolerance of plants.At present,the function of OfNCED3 in Osmanthus fragrans under stress is not clear.In this study,the functions of OfNCED3-1 and OfNCED3-2 were explored by means of subcellular localization,expression pattern analysis as well as transgenic lines of Arabidopsis thaliana.Our study provided theoretical basis for molecular breeding of Osmanthus fragrans.The main results are as follows:(1)Transcriptional regulation analysis of carotenoid metabolism pathway in Osmanthus fragrans1)Quantitative determination of carotenoidsIn this study,petals of ’Yulian Yinsi’(Yingui group,milky white),’Rixiang Guihuang’(Sijigui group,golden yellow),’Dahua Jingui’(Jingui group,orange),’Zhuangyuanhong’(Dangui group,orange-red)are used as materials.Based on HPLC analysis,7 kinds of carotenoids were identified and quantified,and the results showed that the kinds of carotenoids and their contents are different in Osmanthus fragrans varieties.2)Transcriptome sequencing to mine key genes in the carotenoid metabolic pathwayTranscriptomic sequencing analysis of petals of four Osmanthus fragrans cultivars showed that there were 1007,843,365 and 772 differentially expressed genes in ’Yulian Yinsi’,’Rixiang Guihuang’,’Dahuang Jingui’ and ’Zhuangyuan Hong’,respectively.Further Mfuzz co-expression trend analysis and WGCNA analysis showed that OfDXS2,OfCCD1,OfGGPPS2,OfDXR3,OfBCH2 and OfCCD4 were the key genes affecting the accumulation of carotenoids in Osmanthus fragrans petals.3)Prediction of transcription factors(TFs)Transcription factor prediction and Pearson correlation analysis were performed on the promoters of key genes,and as the results,19、16、21、47、21 and 29 TFs with high correlation coefficient(|r|≥0.80)were predicted to potentially bind to the promoters of OfDXS2、OfDXR3、OfCCD1、OfGGPPS2、OfBCH2 and OfCCD4,respectively,suggesting that these TFs may be involved in regulating gene expression and affecting the accumulation of carotenoids in petals.Taken together,our results found the key genes and corresponding transcription factors affecting carotenoid accumulation in Osmanthus fragrans petals,as well as established a regulatory network between key genes and corresponding candidate TFs and provided useful information for further investigation on the potential transcriptional regulation mechanism of carotenoid accumulation in Osmanthus fragrans.(2)Functional study of OfNCED3 gene in Osmanthus fragrans1)Gene cloning,subcellular localization and expression pattern analysis of OfNCED3-1 and OfNCED3-2 in Osmanthus fragrans.In order to investigate the function of OfNCED3 in adapting to stress condition,OfNCED3-1 and OfNCED3-2 genes were successfully cloned from ’Huangchuan Jingui’.OfNCED3-1 and OfNCED3-2 are high homology and both have the unique RPE65 domain of CCD family genes.q RT-PCR analysis found the expression levels of OfNCED3-1 and OfNCED3-2 were higher in leaves and roots,but lower in stems and petals.q RT-PCR analysis found the expression levels of OfNCED3-1 and OfNCED3-2 in leaves were significantly increased after salt stress treatment,both of which increased first and then decreased.transiently overexpressed in tobacco lower epidermal cells found that they are located in the chloroplast,cytoplasm and plasma membrane.2)Identification and functional analysis of transgenic plants of OfNCED3-1 and OfNCED3-2In order to investigate the function of OfNCED3-1 and OfNCED3-2,we successfully transformed OfNCED3-1 and OfNCED3-2 into Arabidopsis thaliana by flower immersion method,and then salt stress was applied to transgenic plants with over-expression OfNCED3-1 and OfNCED3-2.The results showed that the salt tolerance of OfNCED3-2 transgenic plants was higher than that of wild type.In conclusion,our study preliminarily found that OfNCED3-2 plays an important role in plant salt tolerance. |
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