| Tomato fruit contains abundant nutrients and deeply loved by consumers.At the same time,tomato is also the model plant to research fruit development and ripening.Sugars plays an important role in the process of fruit development.At present,lots of sugar transporter family genes have been explored,but the members of the SFP sugar transporter family in tomato have not been reported.Subsequent studies have identified a few other transcription factors,the vast majority of them are transcriptional activators and positively regulate fruit ripening,but the mechanism of is still not clear.In this paper we explores the function of a SFP sugar transporter family gene Sl SFP5 with high expression in tomato fruit development and a MYB transcription factor family gene Sl MYB70,contain an EAR transcription repressor elements.The results are as follows:1.Based on bioinformatics and tissue expression pattern analysis,a sugar transporter protein Sl SFP5 with high expression level in tomato fruit development was analyzed and screened from the sugar transporter family.The promoter of Sl SFP5 was contain many ABA response binding elements ABRE.Transcript accumulation assessed by q RT-PCR indicated that Sl SFP5 can induced by exogenous ABA.It was also found that Sl AREB2 could bind to the promoter of sugar transporter Sl SFP5 gene to regulate the expression level of Sl SFP5.2.Sl SFP5-RNAi transgenic lines were obtained by transgenic technology.The fruit sizes of Sl SFP5-RNAi lines were significantly smaller than wild-type fruits,indicating that Sl SFP5 was involved in fruit development.The content of soluble solids in the transgenic fruit was decreased,and the soluble sugar in the fruit was determined respectively,the content of glucose,sucrose and fructose was decreased,while the content of maltose was not changed.3.The DEGs identified from transcriptome date were involved in plant hormone signal transduction and starch and sucrose metabolic pathways and there were also have lots of genes involved in cell expansion and cell wall synthesis metabolism.A cytochrome b5 gene was selected by screening library by split-ubiquitin yeast two-hybrid system.Through one-to-one verification,it was further confirmed that Sl SFP5 can interaction with Sl CYb5 to affect the fruit development and soluble sugar accumulation.4.Compared and analysised all the genes which contain the EAR motif in the MYB transcription factor family,the gene Sl MYB70 with the highest expression in the fruit was screened.The promoter region of Sl MYB70 contain lots of ethylene responsive binding elements and it can induced by ethylene and ethylene inhibitor 1-MCP.We speculated that it could be participate in ethylene-mediated fruit ripening;It was found that Sl MYB70 have transcriptional inhibitory activity,which was due to the existence of the internal EAR motif,the results indicated that Sl MYB70 functions as a transcriptional repressor.5.Sl MYB70-RNAi and Sl MYB70-OE transgenic plants were obtained by transgenic technology.Sl MYB70-RNAi fruits can accelerated ripening,the content of ethylene and carotenoid was significantly increased than WT;while the Sl MYB70-OE lines were opposite,the fruit ripening was delayed,and the content of ethylene and carotenoid was significantly decreased.Ethylene triple response assay was further proved that Sl MYB70-RNAi lines were sensitive to ethylene in non-fruit tissues.These results demonstrated that Sl MYB70 is required for ethylene-mediated fruit ripening.6.Two ethylene synthesis gene,Sl ACS2 and Sl ACO3,were isolated from transcriptomic data analysis and quantitative PCR verification.The expression level of Sl ACS2 and Sl ACO3 in Sl MYB70-RNAi plants was increased,while in Sl MYB70-OE plants was decreased.Through yeast one-hybrid,EMSA,Ch IP-PCR and dual luciferase reporter system,it was verified in vitro and in vivo that Sl MYB70 can directly bind to the promoters of Sl ACS2 and Sl ACO3 and inhibit the expression of Sl ACS2 and Sl ACO3.In summary,this paper showed that the sugar transporter gene Sl SFP5 can change the tomato fruit size and affect the accumulation of soluble sugar in tomato,as well as that Sl MYB70,act as a transcriptional repressor,regulates the synthesis of ethylene through bind to the promoters of ethylene synthesis genes Sl ACS2 and Sl ACO3.Determintion the function of Sl SFP5 can clarity the regulatory mechanisms of fruit development and sugar accumulation,and also provides a theoretical basis for genetic improvement the flavor quality of tomato fruit.Transcription factor Sl MYB70 negatively regulates fruit ripening have further improved and enriched the ethylene-mediated fruit ripening regulatory network,provide a new type of fruit ripening transcription regulation,and also revealed the regulatory mechanisms of MYB transcription factor and transcription repressor in tomato fruit ripening. |
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