Identification And Functional Analysis Of SWEET Genes In Mulberry

SWEET protein is a new type of sugar transporters identified in recent years.It selectively transports monosaccharides or disaccharides through plasma membrane or intracellular membrane,which plays a key role in plant development and stress response.SWEET protein causes sugar to leak from infected cells,making plants susceptible to pathogens.So far,SWEET genes have been identified in Arabidopsis,rice,tomato,cotton,tobacco,potato,soybean,pear and other plant species.However,few reports about SWEET genes have been reported on mulberry trees and its role in mulberry stress is unknown.In addition,through a transcriptome comparative analysis of the anti mulberry fruit sclerotiniosis varieties and susceptible varieties in our early research,we found that some SWEET genes were closely related to the resistance of this disease.Based on the above considerations,in this study,SWEET genes were fully identified in the mulberry genome database by bioinformatics methods,and its genetic structure,transmembrane domains,and phylogenetic relationship were analyzed.The expression patterns of SWEET genes in green,diseased and red mulberry fruit tissues were analyzed by fluorescence quantitative PCR,and MaSWEET4 and MaSWEET15 were selected as the follow-up research objects.Then,the overexpression vectors of MaSWEET4 and MaSWEET15 were constructed and transferred into tobacco for expression by agrobacterium.Three MaSWEET4 overexpression transgenic tobacco lines and four MaSWEET15 overexpression transgenic tobacco lines were successfully obtained.The expression levels of defense-related genes in positive transgenic lines were detected to explore the mechanism of MaSWEET4 and MaSWEET15 in response to stress.The main research results obtained are as follows: 1.Identification and bioinformatics analysis of SWEET genes in mulberryUsing Arabidopsis SWEET genes as references,15 members of the SWEET gene family were identified in the mulberry genome database.All mulberry SWEET proteins contained two MtN3/Saliva domains and at least one ?-helical transmembrane domain.In the analysis of gene structure and protein motif,mulberry SWEET proteins showed conservation.Most SWEET genes have six exons,and almost all of them have motif1-5.The closely related SWEET proteins also have similar gene structure and motif composition.Based on the prediction of transmembrane domains,the results showed that 11 SWEET proteins own typical 7 ?-helical transmembrane domains,indicating that most of the mulberry SWEET proteins were standard SWEET proteins.In phylogenetic analysis,the SWEET proteins of mulberry and Arabidopsis,rice,and grape were clearly divided into four clades.These four clades correspond to the four well-known clades of Arabidopsis SWEET proteins.2.Analysis of the expression patterns of mulberry SWEET genesThe specific expression pattern of SWEET genes in different fruit tissues of different mulberry varieties was analyzed by fluorescence quantitative technology.The expression leve of each MaSWEET gene varies greatly in different tissues.MaSWEET4 had the highest expression in Jialing 40 diseased mulberry fruit,but almost no expression in red fruit tissue.On the contrary,MaSWEET15 had the hightest expression level in the red fruit of Jialing 40,but almost no expression in the diseased fruit tissue.The expression patterns of MaSWEET4 and MaSWEET15 in diseased fruit and red fruit were also opposite in Jialing 30.In addition,in the red mulberry fruits of three varieties,except MaSWEET15,the expression of most SWEET genes were relatively low.The expression of MaSWEET17 a was highest in green fruit of Morus laevigata wall(Mlw)mulberry,but extremely low expression level in red fruit.Similaryly,the MaSWEET17 a with high expression levels in green fruit of Jialing 40 and Jialing 30,had low expression levels in the red fruit and diseased fruit of the corresponding varieties,indicating that the expression pattern of SWEET genes was similar.3.Transgenic overexpression analysis of mulberry MaSWEET4 and MaSWEET15The overexpression vectors for MaSWEET4 and MaSWEET15 were constructed,and heterologous overexpression in tobacco was performed by leaf disc method.Three strains of MaSWEET4 positive transgenic tobacco and four strains of MaSWEET15 positive transgenic tobacco were obtained.The expression levels of defense-related genes in the positive transgenic lines were detected in the following work.The expression levels of PR2,PDF1.2,ERF1,PI1 and CAT in MaSWEET4 transgenic lines were significantly lower than those of wild type tobacco.Overexpression of MaSWEET15 in tobacco significantly increased the expression levels of PR2 and PR3,and the expression levels of ERF1,PI1,SOD and WRKY12 were also higher than those of wild type tobacco.These results suggested that MaSWEET4 may be a negative regulatory factor and MaSWEET15 may be a positive regulatory factor during the defense response of plant to pathogens.