中国农业科学 ›› 2017, Vol. 50 ›› Issue (16): 3135-3144.doi: 10.3864/j.issn.0578-1752.2017.16.008

• 植物保护 • 上一篇    下一篇

玉米大斑病菌环腺苷酸磷酸二酯酶基因克隆及表达分析

申珅, 李贞杨, 赵玉兰, 李盼, 韩建民, 郝志敏, 董金皋   

  1. 河北农业大学生命科学学院/河北省植物生理与分子病理学重点实验室,河北保定 071001
  • 收稿日期:2017-02-28 出版日期:2017-08-16 发布日期:2017-08-16
  • 通讯作者: 郝志敏,E-mail:hzm_0322@163.com。董金皋,E-mail:dongjingao@126.com
  • 作者简介:申珅,E-mail:shenshen0428@163.com。李贞杨,E-mail:1543639761@qq.com。申珅和李贞杨为同等贡献作者。
  • 基金资助:
    国家自然科学基金(31301616,31601598)、河北省自然科学基金(C2016204160)、河北省高等学校科学技术研究项目(QN2016071)

Cloning and Expression Pattern Analysis of cAMP Phosphodiesterase Coding Genes in Setosphaeria turcica

SHEN Shen, LI ZhenYang, ZHAO YuLan, LI Pan, HAN JianMin, HAO ZhiMin, DONG JinGao   

  1. College of Life Science, Agricultural University of Hebei/Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001, Hebei
  • Received:2017-02-28 Online:2017-08-16 Published:2017-08-16

摘要: 【目的】获得玉米大斑病菌(Setosphaeria turcica)环腺苷酸磷酸二酯酶(cAMP phosphodiesterase, PDE)基因,并分析其在病菌侵染结构发育过程及侵染寄主早期阶段中的表达,为深入探索cAMP信号途径调控病菌致病性的作用机制打下基础。【方法】根据酿酒酵母(Saccharomyces cerevisiae)、白色念珠菌(Candida albicans)、灰霉病菌(Botrytis cinerea)、稻瘟病菌(Magnaporthe oryzae)、绿僵菌(Metarhizium anisopliae)和黑曲霉(Aspergillus niger)6种真菌PDE基因的保守序列,利用简并引物PCR对基因保守片段进行扩增,结合RACE和Genome Walking技术获得基因全长及其侧翼序列;利用MEGA 5.0软件对PDE蛋白预测编码产物进行多重序列比对,并采用邻近法构建系统发育树;利用GSDS分析基因结构,ProtParam分析理化性质,SOMPA预测二级结构,SMART数据库在线分析保守结构域;收集人造疏水介质诱导下侵染结构发育不同时期以及侵染感病寄主叶片不同时间的玉米大斑病菌材料,利用qRT-PCR技术研究StPDE在病菌侵染结构形成过程中不同阶段的转录水平。【结果】玉米大斑病菌基因组中存在1个高亲和力磷酸二酯酶基因(StH-PDE)和1个低亲和力磷酸二酯酶基因(StL-PDE)。其中,StH-PDE全长3 208 bp,包含5个内含子和6个外显子,ORF为2 898 bp。StL-PDE全长5 054 bp,包含4个内含子和5个外显子,ORF为3 090 bp。StH-PDE和StL-PDE分别含有保守的Ⅰ型和Ⅱ型cAMP磷酸二酯酶催化结构域。不同的植物病原真菌中PDE同源基因分别呈现出高度的相似性,其中,StH-PDE与Magnaporthe griseaCordyceps militarisMetarhizium acridum等病原真菌的高亲和力磷酸二酯酶基因聚于同一进化支,StL-PDE与Ascochyta rabiri、Scedosporium apiospermumFusarium oxysporumMetarhizium album等病原真菌的低亲和力磷酸二酯酶基因聚于相同进化支。人造疏水介质诱导下,与菌丝相比,StH-PDE和StL-PDE在分生孢子中的表达水平均显著上调,其中StH-PDE和StL-PDE分别上调了约2倍和52倍。孢子萌发初期两个基因表达水平显著下调,随着萌发的进行,表达水平缓慢回升,至附着胞形成阶段,基因表达出现了第2次高峰,随后表达水平再次下调。在整个过程中,StH-PDE的最高表达水平则出现在附着胞形成时期,达到了菌丝体中的近7倍、分生孢子中的近2倍,而StL-PDE的表达水平始终低于其在分生孢子中的表达水平。StH-PDE和StL-PDE在病菌侵染寄主过程中的表达水平变化趋势与其在人工疏水介质诱导下的表现基本一致。在孢子萌发早期表达显著下调,随着时间延长,缓慢回升,至接种后18和24 h StH-PDE表达水平上调,超过萌发初期。【结论】在病菌侵染结构发育过程中,StH-PDE和StL-PDE均呈现下调-上调-下调的表达水平变化趋势。StH-PDE在附着胞时期表达水平最高,StL-PDE在分生孢子中的相对表达水平最高。

关键词: 玉米大斑病菌, 环腺苷酸磷酸二酯酶, qRT-PCR, 基因表达

Abstract: 【Objective】The objective of this study is to clarify the function of cAMP phosphodiesterase (PDE) and cAMP signal pathway in regulating the pathogenicity of Setosphaeria turcica, the genes encoding cAMP phosphodiesterase were cloned and the expression pattern of these genes were analyzed during the development of infective structures and the early stage of infecting the host. 【Method】Based on conserved sequence of PDE genes from Saccharomyces cerevisiae, Candida albicans, Botrytis cinerea, Magnaporthe oryzae, Metarhizium anisopliae and Aspergillus niger, the homologous fragments were amplified by degenerate primers PCR, and the full length PDE genes and flanking sequences were obtained by combining RACE and Genome Walking. Multiple sequence alignment for prediction coding product of the protein was constructed by MEGA 5.0 software and phylogenetic tree was constructed by adjacent method. Gene structure was analyzed by GSDS. Physical and chemical properties were analyzed by ProtParam. Secondary structure was predicted by SOMPA. Conserved domains were analyzed by SMART on line. The material of different development periods from S. turcica was collected under the induction on artificial hydrophobic medium and on the susceptible host surface. Quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression patterns.【Result】There were one high-affinity phosphodiesterase gene (StH-PDE) and one low-affinity phosphodiesterase gene (StL-PDE) in the genome of S. turcica. Full-length of StH-PDE was 3 208 bp, which consisted of 5 introns and 6 exons, the coding region was 2 898 bp. The full length of StL-PDE was 5 054 bp, which contained 4 introns and 5 exons, with the coding region of 3 090 bp. StH-PDE protein contained conservative type I cAMP phosphodiesterase, and StL-PDE contained L-PDE specific type II cAMP phosphodiesterase catalytic domain. PDE homologous genes in different plant pathogenic fungi presented a high degree of similarity. StH-PDE and the high affinity phosphodiesterase gene from Magnaporthe grisea, Cordyceps militaris, Metarhizium acridum were clustered in the same evolution. StL-PDE and the low affinity phosphodiesterase gene from Ascochyta rabiri, Scedosporium apiospermum, Fusarium oxysporum, Metarhizium album were clustered in the same evolution. Compared with the mycelia, the expression levels of StL-PDE and StH-PDE in conidia were significantly up-regulated, and StL-PDE and StH-PDE were up-regulated by about 52-fold and 2-fold under the induction on artificial hydrophobic medium, respectively. The expression of both genes at the early stage of spore germination was significantly down-regulated. With the germination process, the expression level gradually increased, and the second peak appeared at the appressorial formation stage, and then the expression level was down again. However, in the whole process, the highest expression level of StH-PDE appeared in the appressorial formation period, reaching nearly 7-fold and 2-fold of that in the mycelia and the conidia, respectively, and the expression level of StL-PDE was always lower than that in conidia. During the interaction of S. turcica with the susceptible host, the expression of StH-PDE and StL-PDE in the process of pathogen infection was consistent with that under the induction of artificial hydrophobic material. The expression of both genes in the early stage of spore germination was significantly down-regulated. With the germination process, the expression level gradually increased. The expression of StH-PDE was down-regulated in 18 and 24 h post-inoculation beyond the early stage of spore germination. 【Conclusion】StH-PDE and StL-PDE showed a down-regulation-up-regulated-down-regulated expression pattern in the development of pathogen infection. The relative expression level of StH-PDE was the highest during the appressorial formation, and the expression level of StL-PDE in conidia was the highest.

Key words: Setosphaeria turcica, cAMP phosphodiesterase, quantitative real-time PCR, gene expression