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【目的】克隆小麦条锈菌钙调素依赖蛋白激酶基因Pscamk,并分析其在条锈菌侵染小麦过程中的表达特征及初步功能。【方法】基于本实验室已测序的小麦条锈菌基因组序列,利用RT-PCR方法,从小麦条锈菌生理小种CYR32中克隆Pscamk基因的cDNA序列,并利用网络数据库和生物信息学工具预测该基因编码蛋白的基本特征和保守结构;运用qRT-PCR技术分析Pscamk在不同发育及侵染阶段的表达水平,进一步通过钙调素依赖蛋白激酶(CaMK)的免疫抑制剂KN-93处理小麦条锈菌夏孢子,观察其萌发状况。【结果】获得1个1620 bp的小麦条锈菌CaMK基因Pscamk;序列分析发现,Pscamk编码蛋白包含CaMK蛋白的保守结构域,并与小麦杆锈菌该类蛋白序列相似性最高。qRT-PCR分析表明,Pscamk在条锈菌侵染初期过程中的芽管发育、初生菌丝侵染及吸器形成时期呈显著上调表达,且在条锈菌接种6 h时表达量最高,为对照夏孢子的20.74倍。在专一性免疫抑制剂KN-93处理后,随着KN-93施加浓度的增加,条锈菌夏孢子萌发率逐渐降低,当浓度为1.4μmol/L时夏孢子萌发率为8.02%,仅为对照的12%。【讨论】推测Pscamk基因参与了小麦条锈菌夏孢子萌发、芽管发育以及初期侵染结构的形成。本研究为进一步探索条锈菌细胞钙信号传导机理和致病机制奠定了基础。
【Objective】 The objective of this study was to clone the Pscamk gene from wheat stripe rust and analyze its expression characteristics and primary functions during the process of wheat stripe rust infection. 【Method】 The cDNA sequence of Pscamk gene was cloned from CYR32 of wheat stripe rust genotype by RT-PCR based on the sequenced genomic sequences of wheat stripe rust in our laboratory. The cDNA sequence of Pscamk was predicted by using the web database and bioinformatics tools The gene encodes the basic characteristics and conserved structure of the protein. The qRT-PCR was used to analyze the expression level of Pscamk in different stages of development and infection. The wheat stripe was further treated with the KN-93, an inhibitor of calmodulin-dependent protein kinase (CaMK) Rust spores, to observe the germination. 【Result】 A 1620 bp Pscamk gene of CaMK from wheat stripe rust was obtained. Sequence analysis showed that the protein encoded by Pscamk contained the conserved domain of CaMK protein and had the highest similarity with this protein sequence. qRT-PCR analysis showed that Pscamk was significantly up-regulated during germ tube development, primary mycelium infection and during the formation of the suger during the initial stage of P. rust infection, and was the highest at 6 h 20.74 times of summer spores. After the specific immunosuppressant KN-93 treatment, the spore germination rate of stripe rust decreased with increasing concentration of KN-93, and the spore germination rate was 8.02% at the concentration of 1.4 μmol / L 12% of the control. 【Discussion】 It is speculated that the Pscamk gene is involved in the formation of stripe rust, germ tube development and initial infection structure of wheat stripe rust. This study laid the foundation for further exploration of Ca2 + signaling mechanism and pathogenic mechanism in stripe rust cells.