Deciphering The Regulatory Mechanism Of Biosynthesis Genes Related Steroidal Glycoalkaloids Identified By Genome-Wide Association Study In Tomato

Tomato(Solanum lycopersicum)is one of widely grown horticulture crops in the word and plays a significant role in vegetable industry in our country.At the same time,because of a lot of basic research data released which made the tomato serve as an improtant model plant to study the quality of fruit.Recently,with the improvement of living standards of people,the diet and food safety are becoming increasingly important,which made people set up a healthy,delicaay and safety consumptive idea,which need researcher to continual improving and breeding high-quality tomato.Besides that a lot of studies went on to publish and thanks to the growing important of secondary metabolites,more and more researcher pay great attention to it.Steroidal glycoalkaloids not only is one of main secondary metabolites in tomato,but also is the primary antintutritional due to their toxic effects,so the content of SGA in tomato fruit is an improtant factor which affects the fruit quality.Recently,with the development of metabonomic and sequencing technologies,genome-wide association study coupled with metabolomics has been used more and more to analyze the metabolomic Quantitative Trait Loci(m QTL),up to now more secondary metabolism pathway have been exposed.In this study,we measured the abundance of?-tomatine and its precursor in red ripe stage tomato sampled from 304tomato accession,and then coupled with genome-wide association identified a GAME9which involve in regulating the SGA pathway,moreover,based on its hereditary basis we provide insight into its regulatory function.The main results were shown as follows:1.We determined many SGA-related m QTL in the red fruit of 252 tomato accessionsusing Liquid chromatography-mass spectrometry and two biological repetition wereused in this study,and from these m QTL we verified an GLYCOALKALOIDMETABOLISM 9(GAME9)located in chromosome 1,which is a regulator of SGAproduction.2.Genetic basis of GAME9 analysis in the natural population,the natural variation oftwo functional domains in GAME9 were uncovered:the SNP of 5'N-terminalAP2/ERF domain which lead a alanine(GAME9^135A)to valine(GAME9^135V)substitution have a great impact on the content of hydroxytomatine,and 3'C-terminala serine-rich domain.Biside that we classified GAME9 into four different haplotypesin various tomatoes,namely,GAME9^?,GAME9^?,GAME9^?and GAME9^?.And theSNP of 5'N-terminal AP2/ERF domain may play an important role in regulate SGAproduction.3.The genetic complementation test proved that he SNP of 5'N-terminal AP2/ERFdomain in GAMR9 led its functional alteration.4.Y1H?transient luciferase expression assays and EMSA not only proved GAME9 canbinding with GAME17 promoter but also shown that the binding ability of GAME9depend on its SNP which locate in 5'N-terminal AP2/ERF domain.That is to say,GAME9^?can direct recognize the GAME17 promoter while the GAME9^?can not.5.To analyzing the impact of four different haplotypes of GAME9 on the content ofhydroxytomatine in the natural population and assessing transactivation of fourdifferent haplotypes of GAME9 in the case of the GAME17 promoter,we found thatdifferent haplotypes of GAME9 were used to determine the content ofhydroxytomatine by affecting its binding activity with downstream structural gene.Bisides that natural variation of GAME9 that locate in 3'C-terminal a serine-richdomain also is indispensable domain to exert GAME9 binding activity.6.A novel basic helix-loop-helix(b HLH)factor interact with GAME9 to participate inSGA biosynthesis was confirmed by Y2H?Bi FC and VIGS,further study found that itcan promote the activity of GAME9 in the case of the GAME17 promoter.