Identification And Functionalanalysis Of Bacterial Blight Resistance Genes In Rice

Rice SWEET protein is a novel class of sugar transporter.It can promote the diffusion of carbohydrates from cell membrane into apoplast by cis concentration gradient in cells,and the way is not affected by pH in intracellular environment.The SWEET genes are mainly researched in model plants and food crop,such as Arabidopsis,rice,soybeans,wheat,etc,but the systematic research on SWEET family members in rice is not a lot.In this study,the SWEET gene family was analyzed by bioinformatics analysis of chromosomal location,gene structure,phylogenetic tree and gene evolution,and the SWEET gene induced by bacterial blight strain was screened by gene expression pattern analysis.Then,the mutant of OsSWEET were obtained by CRISPR-Cas9,Observing the pathological changes of the flag leaf after inoculating the bacterial blight strains.qPCR was used to detect the expression of OsSWEET and draft the growth curve of the bacteria.After that,the overexpressed lines were obtained by transgenic technology to verify the effect of the related SWEET gene on plant disease resistance.Finally,the OsSWEET gene is used to expound the interaction between plants and pathogens.Screening out the rice SWEET gene family members in rice database,The OsSWEET members were used to grasp the structural and functional similarities or differences by gene information,chromosome localization,gene structure,amino acid alignment and phylogenetic tree analysis.Amino acid alignment revealed that the SWEET gene family members contain the conserved MtN3 domain,and the domain have multiple phosphorylation sites as their functional regions.The phylogenetic tree analysis found that OsSWEET15,OsSWEET12 and Os8N3,Xa25 and Os11N3,three proteins that have been previously identified as susceptible genes are classified into the same group.Moreover,nucleotide analysis of the family genes revealed that these two genes did not have many effective mutations in the rice domestication process.The inducible analysis of the genes in this cluster screened the OsSWEET12?OsSWEET15 gene induced by some Xanthomonas oryzae pv.oryzae,and the expression level of OsSWEET15 in ZH11 was increased periodically within 24 hours after inoculating with PXO86 strain,reaching a maximum at 24h.The ossweet12 and ossweet15 knockout strains in the background of Zhonghua11 were obtained by CRISPR-Cas9.The knockout strains showed resistant phenotype after inoculation,and the lesion lengths reached 8.04±0.93cm?7.68±0.42cm,respectively,less than WT 12.26±0.94cm.Moreover,qPCR was used to analyze the expression of OsSWEET15 and the growth curve of the bacteria in the knockdown plants T₁ generation after inoculation with PXO86 and PXO79 strains.It was found that the expression of OsSWEET15 in the knockout plants was down-regulated after inoculation with PXO86 and PXO79 strains,and the length of the lesion and the number of Xanthomonas oryzae pv.oryzae is lower than the wild type.The target gene was cloned from Zhonghua 11 by PCR and the OsSWEET15overexpression vector was constructed and transformed into high-resistant indica variety Zhenhui 084.The transgenic seedlings had been obtained by molecular identification.At present,we are cultivating transgenic rice,and then will be transplanted to the field for bacterial inoculation experiments and expression analysis.In summary,Mutations in the OsSWEET15 gene can affect plant resistance to Xoo,Xoo manipulate the sugar transport mechanism of the host plant to increase the diversion of sugars,and affects the survival and reproduction of the pathogen itself by affecting the expression of the plant SWEET gene.OsSWEET15 gene acts as a susceptibility factor in plant disease resistance.The study of the OsSWEET gene laid the foundation for the improvement of the function of the rice SWEET gene family.