中国农业科学2025，Vol.58Issue(18)：3648-3663,16.DOI:10.3864/j.issn.0578-1752.2025.18.006
玉米大斑病菌热激蛋白HSP 9/12基因的克隆与表达分析
Cloning and Expression Analysis of Heat Shock Protein HSP 9/12 Genes in Setosphaeria turcica
摘要
Abstract
[Objective]The objective of this study is to clone HSP 9/12 genes of small heat shock proteins without ACD domain from Setosphaeria turcica and analyze their expression patterns during fungal development,infection,and HT-toxin induction processes.[Method]The coding genes of heat shock protein HSP 9/12 were screened and cloned from the whole genome of S.turcica.Bioinformatics methods were employed to analyze the physicochemical properties,subcellular localization,structural prediction,and phylogenetic analysis of HSP 9/12 proteins.RNA-seq and RT-qPCR were used to examine the expression of HSP 9/12 genes during fungal development,infection,and HT-toxin induction.[Result]Two HSP 9/12 genes were screened and cloned from the S.turcica genome,encoding proteins with 99 and 100 amino acids,respectively.Based on their molecular weights,they were named StHsp10.1 and StHsp10.7.Physicochemical analysis revealed that both HSP 9/12 proteins are hydrophilic,with subcellular localization predictions indicating they are located in the cytoplasm with nuclear localization signals.They lack transmembrane domains and signal peptides,and both contain the HSP9_HSP12(PF04119)domain.StHSP10.1 is an acidic unstable protein,while StHSP10.7 is an alkaline stable protein,both existing predominantly in α-helix-dominated secondary and tertiary structural forms.StHSP10.1 shows closer phylogenetic relationship with Saccharomyces cerevisiae HSP12,whereas StHSP10.7 exhibits closer affinity to Schizosaccharomyces pombe HSP9.The StHSP10.1 exhibited the highest expression during conidial development,followed by hypha,appressoria,and penetration peg,with the lowest expression in germ tubes.After inoculation,the fungal StHSP10.1 expression rapidly increased,reaching 6.37-fold higher FPKM at 72 h compared to 24 h post inoculation.The results of RT-qPCR analysis during the HT-toxin induction process showed that,as the induction time increased,the relative gene expression level of StHSP10.1 in the wild-type strain(WT)significantly increased being 2.9-,14.1-,and 39.8-fold higher at 14,21,and 28 d compared to 7 d,respectively,but remained extremely low in the STK1 gene knockout mutant(ΔSTK1).StHSP10.7 showed extremely low expression levels during fungal development,infection,and HT-toxin induction.AlphaFold 3 predicted that the region from-38 to-24 bp upstream of the transcription start site of the StHSP10.1 contains TATA-box,and binding sites for cell differentiation proteins RCD1 and bZIP transcription factor StbZIP11,simultaneously.Using the STRING online platform to construct the protein-protein interaction network for StHSP10.1,two regulatory pathways of StHSP10.1 were proposed:Ras1→STK1→StbZIP11→StHSP10.1 and Ras1→UBE2→CUE1→RCD1-like→StHSP10.1,suggesting important roles in HT-toxin synthesis and stress induction,respectively.[Conclusion]There are significant differences in the expression patterns of HSP 9/12 genes in S.turcica.StHSP10.1 serves as a key regulatory gene in the processes of pathogen development,infection,and HT-toxin induction,whereas StHSP10.7 has no regulatory effect.关键词
玉米大斑病菌/HSP 9/12/小分子热激蛋白/基因克隆/转录调控Key words
Setosphaeria turcica/HSP 9/12/small heat shock protein(sHSP)/gene cloning/transcriptional regulation引用本文复制引用
张淑红,高凤菊,武秋颖,纪景欣,张运峰,许可,谷守芹,范永山..玉米大斑病菌热激蛋白HSP 9/12基因的克隆与表达分析[J].中国农业科学,2025,58(18):3648-3663,16.基金项目
国家自然科学基金(22078171)、中央引导地方科技发展资金(246Z3610G)、唐山市科技计划(23130221E)、唐山师范学院科研项目(20256129033) （22078171）
