木薯HOS15基因克隆、表达模式分析及原核表达OA

[Objective]The study aimed to clone the highly expressed osmotic stress response factor gene(MeHOS15)of cassava and analyze its expression pattern and prokaryotic expression,thereby providing theoretical references for in-vestigating the regulatory roles of the gene in cassava growth,development,and immune response.[Method]Taking a cassava variety SC124 as the material,the MeHOS15 gene was cloned by PCR and subjected to bioinformatics analysis,and a phylogenetic tree was constructed based on HOS protein sequences from Arabidopsis thaliana and rice.The ex-pression pattern of MeHOS15 under different stress treatments was determined by real-time fluorescence quantitative PCR.The subcellular localization of MeHOS15 protein was observed by constructing pEGAD-MeHOS15-GFP fusion expression vector.The MeHOS15 protein was induced and expressed using a prokaryotic expression system,and expres-sion of the protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Wes-tern blotting.[Result]The MeHOS15 gene was successfully cloned from cassava leaves,with its coding sequence(CDS)of 1725 bp,encoding 574 amino acid,and the cloned gene exhibited a similarity of 99.65％compared with the reference sequence in the Phytozome database(accession number:Manes.12G109000),with its relative protein molecular weight of 64.67 kD and a theoretical isoelectric point of 5.41;MeHOS15 was classified as a stable hydrophilic protein containing the typical conserved the WD40 domain of WD40 protein family.Phylogenetic analysis showed that the MeHOS15 pro-tein had the closest genetic relationship with HOS15 from Arabidopsis thaliana,and they shared similar domain struc-tures.Real-time fluorescence quantitative PCR analysis revealed that the relative expression of MeHOS15 gene exhibited a trend of first increasing and then decreasing under treatments with Xanthomonas axonopodis pv.manihotis(Xam),salt(NaCl,200 μmol/L),hydrogen peroxide(5％H2O2),salicylic acid(SA,50 μmol/L),and abscisic acid(ABA,50 μmol/L).Subcellular localization assays demonstrated that the MeHOS15 protein was localized in the nucleus and cell membrane.SDS-PAGE and Western blotting results confirmed that the protein MeHOS15 was successfully expressed at 28℃.[Conclusion]MeHOS15 is a member of the WD40 gene family,and NaCl,ABA,H2O2,SA,and Xam stresses could induce high expression of the gene,predicting that the MeHOS15 gene is involved in regulating responses of cas-sava to biotic and abiotic stresses.