Function And Molecular Mechanism Analysis Of A Drought Response Gene FMO Gene In Tomato

Plants being sessile are exposed to environmental stress factor naturally during their lifespan,thereby greatly reduce productivity.Abiotic stress is the main environmental stress factor,including drought,salt,cold,heat and waterlogging damage,etc.,which severely restrict the growth and development of plants and then affect the yield and quality of crops.Drought is the main yield restriction factor in plant,and drought caused more crop yield losses than other factors.With the rapid population growth worldwide,food production has become an important topic for us.The potential impact of climate change on crop yields has made this issue even more complicated.At present,more than 70%freshwater resources are consumed for agriculture in the world,and this trend is getting worse under global climate change.Therefore,improving the drought resistance of plants,understanding the molecular mechanism of plant drought stress response,then cultivating drought-resistant crops are of great significance for improving the yield and quality of crops.Tomato is not only one of the most important vegetable crops in the world,but also a fundamental model plant for research.Thus,characterizations of drought response genes,analyzing the detailed function by transgenic research,and exploring the molecular mechanism of drought resistance genes,it will lay a theoretical foundation for the drought resistance improvement for plants.The FMO（FAD-binding Monooxygenase,SGN number:Sopen08g026550） gene is a drought response gene that we isolated form a transcriptome data about tomato differential expression under drought treatment.Bioinformatics and expression analysis show that it response to a various abiotic stresses and hormones.In this study,full-length of FMO was cloned from tomato cDNA.The FMO overexpression vector and an RNAi suppression expression vector were constructed respectively,and they were successfully transferred into drought-sensitive tomato M82（S.lycopersicum cv.M82）through Agrobacterium-mediated transformation.Then,the function of FMO in drought resistance was analyzed,including phenotypic identification,physiological and biochemical changes using transgenic plants.It was shown that inhibiting the expression of FMO can significantly improve the drought tolerance of tomatoes,while overexpression makes them more sensitive.RNA-seq analysis shows that under drought conditions,FMO regulates the expression of a series of drought-tolerant related genes,indicating that FMO may improve the drought tolerance of tomatoes by regulating the expression of drought-related genes.The main findings are as follows:1.Isolation of the FMO gene in tomato and in silico bioinformatics analysisPCR cloned was used to obtain the full-length sequence of tomato FMO.The total length of this gene is 1293bp,encoding 430 amino acids.The results of bioinformatics analysis showed that the FMO gene has high similarity with CTF2A and CTF2B genes in Arabidopsis,and homeopathic elements related to hormones and stress responses are found in the promoter region.Moreover,this gene is expressed in all tissues of common cultivars AC（S.lycopersicum cv.Ailsa Craig）,and highest expression in flowers and fruits,indicating that the drought response gene FMO may have important significance in the later stage of plant growth and development.2.Expression analysis of FMOReal-time PCR was performed to detect and analyze the expression pattern of FMO under hormone ABA and various stress regulators.The results showed that FMO was rapidly down-regulated（1h）under ABA treatment,but returned to normal levels after 6h.After drought and salt treatment,FMO was also rapidly down-regulated（0.25h）,but it was up-regulated again at 0.5h.These indicate that FMO responds quickly at the initial stage of stress or ABA,and it is probably a negative regulator of abiotic stresses.3.Obtained the significantly differential expression of FMO transgenic tomato plantsIn order to study the function of the FMO gene,we constructed the over-expression and RNAi vectors,and obtained over-expression（OE）and RNAi suppressed（Ri）transgenic lines by Agrobacterium-mediated genetic transformation.The over-expression（OE-8,OE-10）and RNAi suppression（Ri-1,Ri-3）transgenic lines with significant expression differences were screened and selected for further functional analysis.4.FMO negatively regulates tomato drought resistanceTransgenic plants with FMO over-expression and silent expression were used as materials for drought stress treatment.Phenotypic identification and physiological and biochemical analysis were carried out to clarify the function of FMO gene in tomato drought resistance.The result was showed that FMO RNAi silent expression transgene improved the drought tolerance compared with wild-type control,while over-expression of FMO is more sensitive.Physiological and biochemical research results showed that RNAi silent expression transgenic plants accumulated more proline（Pro）after drought treatment,and the activities of catalase（CAT）and peroxidase（POD）were higher than those of wild type.Moreover,the relative conductivity and malondialdehyde（MDA）content were lower than wild type and overexpressed line.Tissue staining with DAB and NBT showed that FMO overexpression lines and wild-type plants had more H₂O₂and O^2-enrichment comparing to RNAi lines under drought stress.5.Yeast two-hybrid screening the FMO binding proteinThe FMO as a“bait”to screen the homogenized yeast library of tomato cDNA by yeast two-hybrid,and the genes that interact with it were obtained.A total of 50 yeast positive clones were sequenced,and 18 genes are possible FMO interaction protein,including Catalase（CAT）gene,Ribose diphosphate carboxylase gene,stress reactive protein,etc.The interaction of CAT and FMO was point-to-point validation in yeast.6.RNA-seq analysis of FMO transgenic and wild type plantsThe RNAs in mature leaves of the wild types and the FMO transgenic lines under normal watering control and drought treatment conditions were selected for RNA-seq transcriptome sequencing.Transcriptome analysis results showed that differentially expressed genes（DEGs）increased significantly after drought treatment.The DEGs cluster analysis of shows that many genes expression has changed by the FMO gene（OE/Ri）.Many genes were co-expression with FMO,including PP2Cs,LEA,WRKY,etc.It indicates that FMO regulates the expression of stress resistance-related genes to improve the drought resistance of tomatoes.Enrichment analysis of KEGG pathway showed that FMO genes mainly affect the metabolic pathways related to drought resistance such as plant hormone signal transduction pathway and plant MAPK signal pathway during drought stress.