玉米大斑病菌丝氨酸蛋白酶基因StSp8的克隆、原核表达及其表达模式分析

Cloning,prokaryotic expression and expression pattern analysis of the serine protease gene StSp8 from the Setosphaeria turcica

为了探究玉米大斑病菌丝氨酸蛋白酶基因StSp8的结构特征及其功能,本研究以玉米大斑病菌野生型菌株01-23的cDNA为模版,克隆该基因CDS序列并进行生物信息学分析;构建原核表达载体pET30a-StSp8,在大肠杆菌BL21(DE3)中以IPTG诱导表达目的基因,通过SDS-PAGE技术检测目的蛋白的表达情况;利用qRT-PCR技术检测StSp8基因在不同发育时期和侵染时期的表达量变化。结果表明,丝氨酸蛋白酶StSp8的CDS序列由1602个核苷酸组成,编码533个氨基酸;其编码蛋白包含信号肽结构域、Inhibitor-I9结构域和Peptidase-S8结构域;SDS-PAGE结果显示该蛋白大小为57.18 kD;表达模式分析发现,该基因在病菌发育的菌丝、分生孢子、芽管、附着胞和侵入钉5个典型的发育时期均有表达,其中芽管时期表达量显著升高;StSp8基因在侵染玉米24 h的表达量最高,72 h表达量下降。该研究不仅明确了StSp8基因的结构特征及在病菌生长发育及侵染寄主过程中的表达模式,也为深入揭示其功能奠定了基础。

In order to explore the structural characteristics and functions of the serine protease StSp8 from Setosphaeria turcica,its coding sequence(CDS)was cloned from the cDNA of the wild-type strain 01-23 of S.turcica followed by bioinformatics analysis.The prokaryotic expression vector pET30a-StSp8 was constructed and the target gene was induced by IPTG and expressed in Escherichia coli BL21(DE3).Further,the target protein was confirmed by SDS-PAGE analysis.Moreover,qRT-PCR was employed to investigate the expression level of the StSp8 gene at different developmental stages and infection periods.The results revealed that the CDS of the serine protease StSp8 was consisted of 1602 nucleotides encoding a protein of 533 amino acids.The encoded protein contained a signal peptide domain,an Inhibitor-I9 domain and a peptidase-S8 domain.SDS-PAGE analysis confirmed the molecular weight of the protein was 57.18 kD.Expression analysis showed that the gene was expressed at five typical developmental stages of the pathogen,including mycelium,conidia,germ tube,appressorium and infection peg.It is noteworthy that the gene was significantly highly expressed during the germ tube stage.The expression level of the gene reached its highest level after 24 hours of incubation in maize,followed by a decrease at 72 hours.This study not only clarified the structural characteristics of the StSp8 gene and its expression patterns during the growth,development and infection of the pathogen,but also laid the foundation for further understanding of its functions in S.turcica.