| Rich in terpenoids and flavonoids in its peel and flesh of fruits, tomato is a model material to charactrize the gene functions in primary and secondary metabolisms. Untill now, reports had revealed few transcription factors simultaneously regulating several metabolisms such as carotenoids and flavonoids. However, as an important technology in function analysis of transcription factors, virus-induced gene silencing(VIGS), without genetic transformation involved, has proved itself to be a good complete gene silencing system. Through VIGS, gene expression levels were decreased efficiently and rapidly. Thus, Micro-Tom tomato and the TRV-PDS were used in the study as the material and positive control, respectively; techniques of the VIGS, qRT-PCR, HPLC, GC-MS and LC-MS were used to screen 7 citrus homologous genes. The main research results are as follows:The TRV-PDS positive control and 7 citrus homologous genes TRV-gene vectors with double digestion technology were successfully constructed. The TRV1 / TRV2 virus was detected via PCR amplification from the infiltrated young leaves and fruits. And the newly emerged leaves after infiltration appeared significant photo-bleaching phenomenon resulting from TRV-PDS silencing. The expression levels of all investigated transcription factors gene were significantly decreased in siliencing plants, indicating the TRV could be replicated and transferred efficiently, and played a very good silencing effect.Compared with the control, the total carotenoids in leaves of transcription factors silencing plants were decreased significantly. And the most prominent effect was found in TRV-ABA treatment, with the violaxanthinen decreased by 2.90 times to 0.57 ± 0.06 mg / g(DW); the lutein decreased by 2.25 times to 3.85 ± 0.22 mg / g(DW); the β- carotenoid, zeaxanthin and lycopene decreased by 2.14, 3.80 and 2.59 times, respectively. In addition, zeaxanthin and lycopene in leaves of TRV-RAP and TRV-AGL silencing plants were also significant decreased by nearly 6 and 3 times, respectively.Compared with the control, the total carotenoids in tomato fruits of silencing plant were incresed. Although the β- carotene greatly decreased to 21.64 ± 1.18 μg / g(FW) in TRV-ACO silencing fruits, the lycopene and γ- carotene were increased by 1.90, 2.53 times, respectively, in TRV-RAP silencing plants v.s. the control; the γ- carotene was also increased by 2 times among TRV-ARG, TRV-DET, TRV-ABA, TRV-GA silencing fruits.By using LC-MS technology, 6 amino acids, 26 phenolic acids and flavonoids were detected in Micro-Tom tomato leaves, with the most abundant metabolite as the α-tomatine, reaching 13.54 ± 2.91 μg / g(DW). Compared with the control, as revealed by MPP analysis, flavonoids profiles kept relatively stable among treatmetns and the control.However, complicated profiles of 11 amino acids, 43 phenolic acids and flavonoids were detected in Micro-Tom tomato fruit with LC-MS technology, with the dominant metabolite as cumaric acid, reaching 22.26 ± 1.77 μg / g(DW); fold changes of several metabolites were found between fruits of silencing plants and the control, such as kaempferol-3-O-glycoside, rutin, α-tomatine, caffeoylputrescine etc.By using GC-MS, 38 volatiles were detected in tomato fruits, with aldehydes were dominant. Notably, the highest content was found with 3-hexanal in TRV-DET silencing fruits, reaching 13.89 ± 0.93 μg / g(FW). After TRV-gene silencing, most of the volatiles presented significant changes, such as 3-hexanal, trans-2-hexenal, α- pinene, etc.In conclusion, it suggested that the investigated citrus homologous genes regulate the metabolic pathways of terpenoids and other metabolisms in the tomato fruits simultanously. |
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