| Ocimene is one of the main fragrance components of Hedychium coronarium.The synthesis and release of ocimene are affected by many factors,and plant hormones play an important role in them.Ethylene as a gas phytohormones participates in many stress responses in plants,and it is closely related to the flowering of plants and the synthesis and release of fragrance substances.In this study,Hedychium coronarium was used as the research object.The relationship between ethylene and the release of ocimene was explored by treatments with exogenous ethylene and ethylene inhibitor 1-MCP,GC-MS analysis of Hedychium coronarium at different development stages,and RT-qPCR analysis.RT-PCR was used to clone two EIN3/EIL1 family members HcEIL1-1 and HcEIL1-2 from flowers of Hedychium coronarium.The regulation effects of HcEIL1-1 and HcEIL1-2 on terpenoids release,related terpene synthase genes and transcription factor HcMYC2-1 were investigated by Viral silencing,RT-qPCR,yeast two-hybrid,two-molecule fluorescence complementation,and subcellular localization.And yeast single-hybrid and dual-luciferase techniques were used to explore the regulatory relationship between HcEIL1-1/2 and HcTPS3 promoter.This study lays a theoretical foundation for exploring the molecular mechanism of the formation of terpenoids and the ethylene signaling pathway to regulate the synthesis and release of fragrance substances in Hedychium coronarium.The main results are as follows:1,Two EIN3/EIL1 family member genes were cloned from the cDNA of Hedychium coronarium flowers,by using RT-PCR technology,named HcEIL1-1 and HcEIL1-2,respectively.Sequence analysis revealed that the maximum read length of HcEIL1-1 is 1863 bp,which encodes 620 amino acids.The maximum read length of HcEIL1-2 is 1848 bp,which encodes 615 amino acids.Phylogenetic tree analysis showed that HcEIL1-1 and HcEIL1-2 clustered respectively together with MaEIL1-1 and MaEIL1-2,but they were far apart,suggesting that the functions are different among them.Amino acid sequence analysis revealed that both HcEIL1-1 and HcEIL1-2 have conserved sequences and characteristic regions of EIN3/EIL1,an acidic amino acid aggregation region(AD),a proline-rich region(PR),and five basic amino acids aggregation area(BDI-V).2,Through the use of exogenous ethylene and 1-MCP treatment of Hedychium coronarium,the dynamic changes of the release of ocimene at different development stages were determined.The results showed that the release of ocimene after ethylene treatment was higher than that of the control group,while the release of ocimene after 1-MCP treatment was lower than that of the control group.Combined with RT-qPCR analysis,it was found that compared with the control group,ethylene could increase the expression of HcEIL1-1,HcEIL1-2 and HcTPS3 at different development stages,and 1-MCP inhibited the expression of these genes,and the expression pattern of HcTPS3 was similar to that of HcEIL1-1 and HcEIL1-2.3,After transient virus silencing HcEIL1-1/2 of the petals of Hedychium coronarium,The content of the terpene volatiles was determined by GC-MS technique.The results showed that: after HcEIL1-1 was silenced,ocimene,linalool,and allo-ocimene decreased remarkably by 33 %,37 %,and 47 %.After HcEIL1-2 was silenced,ocimene,linalool,and allo-ocimene decreased remarkably by 33 %,42 %,and 49 %.Combined with RT-qPCR analysis,after HcEIL1-1 was silenced,the expression of HcEIL1-1 decreased by 46 %,and the expression levels of HcMYC2-1,HcTPS1,HcTPS3,and HcTPS10 decreased respectively by 53 %,53 %,40 %,and 50 %;After HcEIL1-2 was silenced,the expression of HcEIL1-2 decreased by 70 %,and the expression levels of HcMYC2-1,HcTPS1,HcTPS3,and HcTPS10 decreased respectively by 15 %,13 %,42 %,and 13 %.4,The full-length gene fragments of HcEIL1-1 and HcEIL1-2 were constructed on the p35S-GFP vector,respectively.Through PEG-mediated Arabidopsis protoplasts found that HcEIL1-1 and HcEIL1-2 were localized in the nucleus.5,Yeast two-hybrid experiments were performed and the results showed that HcEIL1-1 and HcEIL1-2 could interact with HcMYC2-1.In order to eliminate the false-positive combination of yeast two-hybrids,two-molecule fluorescence complementation experiments were performed.The results showed that only HcEIL1-1 could interact with HcMYC2-1.6,Yeast one-hybrid experiments were performed and the results showed that HcEIL1-1 could bind to the HcTPS3 promoter.A further dual luciferase assay revealed that both HcEIL1-1 and HcEIL1-2 can bind to the HcTPS3 promoter,Compared with the control,the LUC/REN fluorescence ratio of pGreen-II-62-SK-HcEIL1-1+HcTPS3-LUC and pGreen-II-62-SK-HcEIL1-2+HcTPS3-LUC was increased by 4.75 and 3.63 times,respectively. |
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