Functional Study And Application Of Tomato Fusarium Wilt Resistance Gene SlySGT1

As a vegetable crop rich in nutrients,tomato(Solanum lycopersicum)has extremely high value and is deeply loved by people.It has a wide distribution area and is widely cultivated globally.Tomato wilt disease is a soil borne ungal disease caused by Fusarium oxysporum f.sp.Lycopersici(Fol).Fusarium oxysporum belongs to a group of facultative parasitic fungi,which have subspecific differentiation and multiple specialized forms,and can simultaneously infect host plants and survive in the soil,causing serious economic losses to agricultural production.The prevention and control of tomato wilt disease is difficult.Currently,in agricultural production,selecting and utilizing resistant varieties is the main control method for tomato wilt disease.However,conventional breeding takes a long time and has low efficiency.However,using molecular breeding technology can accurately and quickly improve breeding efficiency.Therefore,in-depth exploration of the disease resistance mechanism of tomato wilt disease and the cultivation of new resistant varieties of tomato using molecular breeding technology have become a new direction for tomato wilt disease prevention and control,It is of great significance.Our previous results showed that after Fol infected tomato resistance strain Motelle,the expression level of endogenous sRNA in some tomatoes changed.The expression of Sly-miR482e-3p is inhibited,and the knockdown of Sly-miR482e-3p not only affects the expression of two NBS-LRR target genes,but also changes the expression of genes related to plant hormone ethylene signaling pathway,which is involved in tomato resistance to Fusarium oxysporum through some mechanism.SGT1 was also predicted to be the target gene of Sly-miR482e-3p.SGT1 is a recently discovered gene related to plant disease resistance regulation.Its function can regulate the assembly of centromere and the modification of target proteins by ubiquitin.In order to investigate the biological function of SGT1 in tomato fusarium wilt resistance.The findings of the study are as follows:1.The bioinformatics prediction analysis of SlySGT1 shows that SlySGT1 has no signal peptide and is not a secreted protein;SlySGT1 does not have a transmembrane domain and is not a membrane protein;SGT1 is located in the nucleus of tomato cells.2.By using subcellular localization techniques,the localization of SGT1 in tomato overexpressing transgenic material cells was observed.The results showed that SlySGT1 was located in the nucleus,and the actual results were consistent with the predicted results from previous websites.3.Using CRISPR/Cas9 gene knockout and overexpression techniques,SlySGT1 was overexpressed in the susceptible variety Moneymaker(MM)through Agrobacterium mediated tomato genetic transformation.SlySGT1 was knocked out in the resistant variety Motelle(Mot),and four transgenic overexpressed plants,Linel,Line2,Line3,and Line4,were obtained,providing research materials for subsequent research on the disease resistance of SlySGT1.4.The results of qRT-PCR showed that SlySGT1 expression levels of the four overexpressed plants were significantly higher than those of the tomato susceptible variety Moneymaker.The results of resistance analysis of four overexpressed transgenic plants under specific Fusarium oxysporum tomato infection showed that SlySGT1 overexpression in susceptible variety Moneymaker resulted in increased resistance to pathogen Fol,indicating that SlySGT1 was involved in tomato Fusarium wilt resistance process.5.SGT1 is involved in ethylene signal transduction pathway.In order to explore the interaction mechanism between SGT1 and key genes in the ethylene signal pathway,Using yeast two-hybrid technology,the Mating library was screened,and the sequencing results were compared with the Blast function in the NCBI database,and 20 candidate proteins that interacted with SlySGT1 were obtained.6.The candidate SlySGT1 proteins were identified by yeast point-to-point rotation verification、BiFC,and the results showed that Solyc04g009440、Solyc09g080660、Solyc09g031930 interacted with SlySGT1.In conclusion,this study provides a theoretical basis for further analysis of SlySGT1’s role in tomato fusarium wilt resistance,proving that SlySGT1 plays a certain positive regulatory role in tomato growth and development,and participates in tomato fusarium wilt resistance.It provides a useful direction for developing new tomato varieties resistant to fusarium wilt and exploring molecular genetic breeding techniques.