| Fragrance is one of the most important factors determining the quality of tobacco(Nicotiana tabacum) leaves. This study is aimed to identify the five fragrant mutants(fgr1-fgr5) acquired through ethyl methane sulfonate(EMS) mutagenesis of Zhongyan100, an elite tobacco cultivar. The middle leaves of Zhongyan-100 and the mutants were obtained to calculate the trichome densities. A portable electronic nose PEN3 was used to detect the fragrant compounds of fresh leaves and flue-cured leaves of Zhongyan-100 and the mutants, and principal component analysis and cluster analysis were performed with the response value of the e-nose. Suppression subtraction hybridization(SSH) was further performed between two typical mutants and Zhongyan-100, and fragrance related genes were chose from SSH libraries. Real-time quantitative PCR were performed to study the expression of fragrance related genes. The main results were list as follows:(1) The fully-expanding middle leaves of Zhongyan-100 and the mutants were obtained to calculate the trichome densities at five different development stages: fast-growing stage, squaring stage, full-blossom stage, early-ripening stage, late-ripening stage. Compared with Zhongyan-100, these five mutants showed distinct patterns of leaf maturity and higher glandular trichome densities of leaf surface, both of which were thought to be contributed greatly to tobacco flavor. The mutants fgr3 and fgr4 had the most highly trichome density.(2) The portable e-nose PEN3 was used to detect the fragrant compounds of fresh leaves and flue-cured leaves of Zhongyan-100 and the mutants. The difference between the e-nose response curve of fresh leaves and flue-cured leaves suggested that the fragrant compounds changed greatly at the ripening and curing process. From the radar response figure of the e-nose we found that the response to fresh leaves showed varied patterns between different mutants. According to the results of principal component analysis and cluster analysis, the mutants can be divided into two groups: “sun-cured tobacco†and “flue-cured tobaccoâ€.(3) Two forward and two reverse SSH libraries were constructed between “sun-cured tobacco†fgr3, “flue-cured tobacco†fgr4 and Zhongyan-100. A total of 1536 positive colonies were randomly picked for sequencing and 1017 high quality ESTs were acquired which were assembled into 202 unigenes. Based on the results of gene annotation and GO analysis, we found 12 of the 202 unigenes probably involved in fragrance metabolism.(4) We selected other 19 genes that were reported to be closely related to fragrance metabolism according to published literatures. All the expressions of 31 genes were analyzed by quantitative real-time PCR(q PCR) with the upper leaves of Zhongyan-100 and its mutants fgr3 and fgr4 at five different development stages. From the results of q PCR 14 genes were confirmed to contribute greatly to fragrance of the mutants, which mainly involved in phenylpropanoid-flavonoid pathway, MEP-carotenoid pathway and duvatrienediol biosynthesis pathway. DXR was highly expressed in fgr3 compared with Zhongyan-100 at the first three development stages, which suggested that MEP pathway contributed greatly to high level fragrance of fgr3; the expression of PSY was significant high in fgr4 compared with Zhongyan-100 at squaring stage and full-blossom stage, indicating an important role of carotenoid pathway in fgr4. The different patterns of gene expression between the two mutants indicated that they might have different mechanisms of regulating fragrance related genes.(5)The trichome density of fgr3 and fgr4 was significantly higher than Zhongyan-100, and DXR was highly expressed in fgr3 and the expression of PSY was significant high in fgr4 compared with Zhongyan-100, which identified that fgr3 and fgr4 were fragrance-enriched mutants. |
