摘要
【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] To clone calcium-dependent protein kinase gene （camk） from Puccinia striiformis f. sp. tritici （Pst） and analyze its function. [ Methods ] The cDNA full-length of Pscamk was isolated by using reverse transcriptional- PCR （RT-PCR） , and gene expression profile at different morphological stages was analyzed via quantitative real-time - PCR（qRT-PCR）. Pst urediospores were treated with CaMK suppressor KN-93 and germination rate was investigated. [ Results] A gene cDNA full-length with ! 620 bp was obtained and designated as Pscamk. qRT-PCR analysis showed Pscamk expression was highly induced in the early stages of Pst infection and reached the maximum at 6 h post inoculation （hpi） as 20.74-fold as that in the control （0 hpi）. With increasing of the concentration of CaMK suppressor KN-93, germination rate of Pst urediospores was gradually decreased. The germination rate was reduced to 8.02% , only 12% of the control, under 1.4 μmol/L KN-93 treatment at 10 h after incubation at 9℃. [ Conclusion] Pscamk might play a role in germination and germ tube elongation of Pst urediospores. This study provides a basis for exploring pathogenesis of calcium signaling pathway during Pst infection.
出处
《微生物学报》
CAS
CSCD
北大核心
2014年第11期1296-1303,共8页
Acta Microbiologica Sinica
基金
国家"973项目"--国家重点基础研究发展计划(2013CB127700)
国家自然科学基金项目(31371889
31171795)
高等学校学科创新引智计划资助项目(B07049)
教育部新世纪优秀人才支持计划(NCET-12-0471)
西北农林科技大学基本科研业务费优青项目(YQ2013001)~~
