摘要：

碱性亮氨酸拉链(bZIP)转录因子蛋白是一类在动植物及微生物中结构和功能均具有保守性的转录调控因子,为明确bZIP类转录因子在植物病原真菌中的功能及作用机制,进一步确定其与病菌生长发育及致病性的关系,以玉米大斑病菌01-23为材料克隆得到了StbZIP9基因(GenBank No.XM_008032179.1),StbZIP9是bZIP转录因子家族成员之一,对该基因结构及蛋白质特征分析结果显示,该基因全长788 bp,开放阅读框为726 bp,编码241个氨基酸,其编码蛋白包含一个在真菌中高度保守的同源结构域BRLZ;对该基因在病菌生长发育及侵染宿主过程的RNA-seq数据进行分析,发现与菌丝时期相比StbZIP9在附着胞和芽管时期表达量升高2~4倍,在侵染玉米叶片24,72 h后基因表达从无到有且持续升高,表明StbZIP9与附着胞发育和芽管形成相关联并在病菌侵染宿主细胞过程中发挥重要作用;进一步利用生物信息学技术预测其结合保守基序及调控的靶基因,结合基序为NNTWACGTNN,包含bZIP 类转录因子识别核心序列ACGT,并根据该序列预测StbZIP9下游靶基因,结合RNA-seq数据进行表达模式分析得到4个下游靶基因(JGI数据库中蛋白ID分别为:132893、163024、162798、40466),功能注释表明,其参与细胞壁组分聚合和运输、侵染宿主、孢子休眠等多种生物过程。为进一步阐明其在参与调控病菌侵染寄主的分子机制提供依据。

关键词: 玉米大斑病菌, 转录因子, 基因克隆, 表达模式分析, 靶基因筛选

Abstract:

Basic leucine zipper(bZIP)transcription factor protein is a kind of transcription factor with conservative structure and function in animals,plants and microorganisms.In order to clarify the function and mechanism of bZIP transcription factor in plant pathogenic fungi,and further determine its relationship with the growth,development and pathogenicity of the pathogen,the StbZIP9 gene was cloned from Setosphaeria turcica 01-23(GenBank No.XM _ 008032179.1).StbZIP9 is a member of the bZIP transcription factor family.The analysis of the gene structure and protein characteristics showed that the DNA sequence was 788 bp in length,with an open reading frame of 726 bp,encoding 241 amino acids.The encoded protein contained a highly conserved homologous domain BRLZ in fungi.The RNA-seq data of the gene during the growth and development of the pathogen and the process of infecting the host were analyzed.It was found that the expression level of StbZIP9 was 2 to 4 times higher than that in the appressorium and germ tube period compared with the mycelium period.After 24,72 h of infection of maize leaves,the gene expression increased from scratch and continued to increase,indicating that StbZIP9 was associated with appressorium development and germ tube formation and played an important role in the process of pathogen infecting host cells.Further,bioinformatics techniques were used to predict its binding conserved motifs and regulatory target genes.The binding motif was NNTWACGTNN,including the bZIP transcription factor recognition core sequence ACGT,and the downstream target genes of StbZIP9 were predicted according to the sequence.Combined with the expression pattern analysis using the RNA-seq data,four downstream target genes(protein IDs in the JGI database were :132893,163024,162798,40466)were obtained,and the functional annotation table was obtained.The functional annotation revealed its involvement in many biological processes, such as polymerization and transport of cell wall components, host infection, and spore dormancy. It will provide the basis for further elucidation of the molecular mechanism involved in the regulation of pathogen infection.

Key words: Setosphaeria turcica, Transcription factors, Gene cloning, Expression pattern analysis, Target gene screening