芸薹根肿菌分泌蛋白PbSP1的克隆及表达特征分析

Characterization and Expression Profiling of PbSP1 in Plasmodiophora brassicae

【目的】对芸薹根肿菌PbSP1进行克隆和功能分析,丰富芸薹根肿菌分泌蛋白的科学认知。【方法】通过生物信息学分析、表达动态变化和异源表达对芸薹根肿菌分泌蛋白PbSP1进行研究。【结果】通过测序分析发现,PbSP1的序列与已经公布的芸薹根肿菌e3菌株的参考基因组对应基因序列一致,且不存在内含子。生物信息学分析发现PbSP1氨基酸序列的氨基端存在一段信号肽序列,同时序列中存在4个Kazal结构域。通过酵母蔗糖酶分泌实验进一步发现,PbSP1编码的信号肽序列具有活性。通过不同阶段表达特征分析发现,PbSP1在不同时期差异表达,其中在芸薹根肿菌休眠孢子萌发期表达最为活跃。通过亚细胞定位实验发现PbSP1定位在细胞质内。【结论】PbSP1可以被芸薹根肿菌分泌到胞外,并在芸薹根肿菌休眠孢子萌发过程中发挥重要功能。

【Objective】The current study aimed to characterize the PbSP1 of Plasmodiophora brassicae,and enrich our knowledge of secreted proteins in P.brassicae.【Method】Bioinformatic analysis combined with expression profiling and heterologous expression was employed to characterize a putative secreted protein PbSP1 in Plasmodiophora brassicae.【Result】Sequencing analysis revealed that DNA sequence of Pb-SP1 was identical to that in the reference genome of e3 strain with no intron.Bioinformatic analysis of the amino acid sequence of PbSP1 showed that there was a signal peptide sequence(1-19 aa)at the N-terminus of PbSP1,and there were 4 Kazal domains in the sequence.Furthermore,the presence of functional signal peptide was verified by yeast invertase secretion assay.Expression profiling revealed that Pb-SP1 was differentially expressed at different stages,which was the most active during the resting spore germination.Transient expression of PbSP1 in tobacco showed that PbSP1 was distributed in the cytoplasm.【Conclusion】The results above suggest that PbSP1 can be secreted extracellularly and play an important role during germination of resting spores in P.brassicae.