木薯糖转运蛋白MeSWEET10-8基因克隆与功能分析OA

[Objective]The sugar transporter protein MeSWEET10-8 gene of cassava was cloned,and its function was analyzed to provide theoretical reference for exploring the sugar transport regulation mechanism of MeSWEET10-8 gene and its interaction function with plant pathogen.[Method]Using the cassava variety Huanan 9 as the research material,with the cDNA of cassava mature leaves inoculated with Xanthomonas axonopodis pv.manihotis(Xam)as the template,the MeSWEET10-8 gene was amplified by PCR.Its bioinformatics analysis was conducted,and the phylogenetic tree of SWEET proteins in different species was constructed using MEGA 11.0.The gene function was analyzed through subcellu-lar localization,sugar transport experiments,and transcriptome sequencing(RNA-seq).The effects of inoculation with Xam on the expression of MeSWEET10-8 gene was detected by real-time fluorescence quantitative PCR.[Result]The coding sequence(CDS)of MeSWEET10-8 gene was cloned from the mature diseased leaves of cassava.The length of this sequence was 840 bp,which was exactly the same as the reference sequence(accession number:Manes.14G047700)in the Phytozome database.The encoded protein had a molecular weight of 31.81 kD,a theoretical isoelectric point(pI)of 8.18,and did not contain signal peptide.MeSWEET10-8 protein contained 2 conserved MtN3_Slv functional domains and 7 transmembrane domains.The promoter region contained five light-responsive elements(Box 4,G-box,GA-motif,GT1-motif,and TCT-motif),one salicylic acid(SA)responsive element(TCA-element),one abscisic acid(ABA)responsive element(ABRE),one methyl jasmonate(MeJA)responsive element(TGACG-motif),two gibberellin(GA)responsive elements(CGTCA-motif,GARE-motif),and three biological and abiotic stress response elements(ARE,LTR,TC-rich repeats).Phylogenetic analysis showed that MeSWEET10-8 protein had the closest genetic rela-tionship with the AtSWEET10 of Arabidopsis thaliana,it located in the Clade III with pathogenic OsSWEET13 and OsS-WEET14 proteins.Subcellular localization and sugar transport assay demonstrated that the MeSWEET10-8 protein was lo-cated on the cell membrane and had the function of transporting xylose.RNA-seq results showed that the MeSWEET10-8 gene was highly expressed in the stem tissues of cassava,followed by the petiole and root apical meristems,and no expression was detected in the leaves and storage roots.Real-time fluorescence quantitative PCR results indicated that MeSWEET10-8 gene was induced by Xam,and its relative expression gradually increased in the early stage of inocula-tion,reaching the highest value at 72 h,and then began to decline.[Conclusion]The cassava MeSWEET10-8 protein functions in transporting xyloses and is induced during Xam infection,indicating that MeSWEET10-8 protein is involved in the interaction between plants and pathogens,and may play a significant physiological role in plant stem development.