Cloning and functional analysis of cassava sugar transporter protein MeSWEET10-8 gene

【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 subcellular 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 relationship with the AtSWEET10 of Arabidopsis thaliana， it located in the Clade III with pathogenic OsSWEET13 and OsSWEET14 proteins. Subcellular localization and sugar transport assay demonstrated that the MeSWEET10-8 protein was located 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 inoculation， 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.