Cloning and Expression Analysis of Genes of Small Heat Shock Protein in Setosphaeria turcica
[Objective]The objective of this study is to clone the small heat shock protein(sHSP)genes in Setosphaeria turcica,elucidate their structural characteristics,and explore their expression profiles during pathogen development and in response to HT-toxin induction.[Method]The hidden Markov model(HMM)was used to identify sHSP family members across the entire S.turcica genome.PCR technology was used to clone sHSP genes from S.turcica strain 01-23.Bioinformatics methods were then applied for the analysis of physicochemical properties,subcellular localization,structural prediction,and phylogenetic analysis of the sHSP genes obtained.Additionally,RNA-Seq and RT-qPCR were performed to determine the expression of sHSP genes across different developmental stages and during HT-toxin induction in S.turcica.[Result]Three sHSP family members(StHSP37.2,StHSP37.0 and StHSP22.6)were identified from the genome of S.turcica.The corresponding DNA sequences were successfully cloned from strain 01-23.The encoded sHSP proteins were weakly acidic and hydrophilic proteins,without transmembrane domain or signal peptide.Random coil in the secondary structure accounted for 58.97%to 60.35%,and β-turn ranged from 2.69%to 7.83%only.Subcellular localization prediction indicated that StHSP37.2 and StHSP37.0 were located in the nucleus,while StHSP22.6 was located in both nucleus and cytoplasm.Conserved ACD_sHSP-like domains were identified near C-terminus,with 2,3,and 5 conserved motifs in StHSP37.2,StHSP37.0,and StHSP22.6,respectively.The monomer tertiary structure models of sHSP were constructed using SWISS-Model and AlphaFill.Phylogenetic analysis indicated close relationships between StHSP22.6 and sHSP in Alternaria alternata,and between StHSP37.2/StHSP37.0 and sHSP in Bipolaris maydis.The sHSP genes of S.turcica had the highest expression levels in hyphae,followed by germ tubes,appressoria,and penetration pegs,with the lowest expression levels in conidia.StHSP22.6 and StHSP37.2 showed significant negative correlations with HT-toxin induction,and the relative gene expression was upregulated by 6.45 and 18.12 folds on day 14,respectively.On day 21 and 28,StHSP37.2 showed modest upregulations of 2.56 and 1.78 folds,respectively,while StHSP22.6 did not differ from the wild-type(WT).StHSP37.0 exhibited significant positive correlations with HT-toxin induction,with a significant downregulation by 59.23%,86.30%,and 88.11%on day 14,21,and 28,respectively.Exploration of expressed genes significantly associated with sHSP of S.turcica suggested that StHSP37.2 and StHSP22.6 were mainly related to HSP90,HSP104,catabolism,and mitochondrial Mg2+transport,while StHSP37.0 appeared to be associated with vacuolar alkaline amino acid transport,organic synthesis,and substance secretion.[Conclusion]The sHSP family members in S.turcica demonstrate a high degree of conservation yet exhibit structural and phylogenetic differences from other sHSPs.They are integral to the development of hyphae,germ tubes,appressoria,and penetration pegs,and also exert significant regulatory functions during HT-toxin induction.
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