Function Verification Of Polyphenol-related Transcription Factors From Tomato By Transient Expression System

Polyphenols are very common antioxidants in daily life and are an important part of plant secondary metabolites.Luo et al.(2008)found that AtMYB12 gene could regulate the synthesis of flavonoids in transgenic tomato fruits,and could stimulate the synthesis of downstream metabolites caffeoyl quinic acid.In tobacco,the AtMYB12 gene only stimulates the synthesis of flavonoids.Butelli et al.(2008)transferred the two anthocyanin synthesis regulators,Delila(bHLH transcription factor)and Rosea1(MYB transcription factor),into tomatoes,which resulted in various polyphenols in tomato fruits,especially anthocyanins and other flavonoids content increased significantly.Tobacco is a model crop in transgenic research,and has formed a complete set of more mature technical processes.So tobacco can be used as a good expression system to quickly verify the function of genes.In our laboratory,transcriptome sequencing and comparative analysis were performed.We found that some transcription factors were associated with enrichment of flavonols in tomatoes caused by the AtMYB12 gene,including 22 transcription factors,such as MYB,WD-40 and bHLH.In order to explore the role of these transcription factors in the regulation of these polyphenols,this study has carried out a preliminary study on its function.In order to facilitate the preliminary functional analysis of candidate transcription factors,the expression of transient expression system was established by using pGR106 and pRI101 as expression vectors with AtMYB12 transcription factors,and the optimal expression system was established and could be used for gene regulation rapid functional analysis of secondary metabolites synthesis.The results showed that these four transcription factors,SlMYB1 ?SlbHLH96?WD40-19?SlbHLH072,could cause the change of the content of polyphenols,and the first three were positive,the latter play a negative regulatory role.In addition,when the expression of ROS1 was transiently expressed,the accumulation of anthocyanins was found in tobacco leaves at about 4 days.When the co-expression of Sl MYB1 and ROS1 was observed,the anthocyanin accumulation of tobacco leaves was also found,but weaker than that of ROS1 expressed separately,indicating that SlMYB1 may inhibits the synthesis of anthocyanins by ROS1.In this study,the transient expression system was successfully used in tobacco to verifythe related candidate transcription factors for polyphenols.The successful development of this system provides a simple and easy way to identify key candidate transcription factors from a large number of transcriptome sequencing data,and then greatly shortening the cumbersome work brought about by the steady genetic expression.So it can provide an important research basis for the subsequent genetic transformation of tomato to verify the function of candidate transcription factors and to cultivate tomato germplasm of high polyphenol content.