小麦条锈菌效应蛋白基因PSTG_23616的时空表达特征分析

Spatial and Temporal Expression Pattern of Effector Protein Gene PSTG_23616 in Puccinia striiformis f.sp. tritici

明确条锈菌（Puccinia striiformis f.sp.tritici,Pst）效应蛋白基因PSTG_23616的时空表达特征,旨在解析该基因在病菌生长发育及致病过程中的作用。利用RT-PCR和PCR克隆获得PSTG-23616的cDNA和基因组DNA全长;借助生物信息学软件分析PSTG-23616蛋白序列特征并构建系统进化树;通过酵母分泌系统、农杆菌介导的瞬时表达系统、实时荧光定量RT-PCR（qRT-PCR）、亚细胞定位技术初步明确该基因的功能。结果表明,PSTG_23616基因组全长937bp,开放阅读框（open reading frame,ORF）全长699bp。生物信息学软件预测结果显示,PSTG-23616信号肽为位于N端的1-22个氨基酸;酵母分泌系统分析表明,PSTG-23616的信号肽具有分泌功能,为典型的分泌蛋白;注射含有PSTG_23616的重组农杆菌菌液,可抑制由BAX引起的细胞程序性死亡（Programmed cell death,PCD）反应;qRT-PCR结果表明,PSTG_23616在小麦条锈菌初侵染过程中一直上调表达,24h表达量达到顶峰;亚细胞定位分析表明,PSTG_23616在整个细胞均有分布。推测,该效应蛋白参与条锈菌侵染结构的分化,同时在条锈菌与小麦互作过程中发挥毒性功能。

In this study, we characterized spatial and temporal expression pattern of Pst （Puccinia striiformis f. sp. tritici） effeetor protein PSTG_23616 for further functional characterization of PSTG _23616. The full-length of eDNA and DNA sequences of PSTG_23GJ6 were obtained by RT-PCR and PCR technology. Bioinformatie methods were used to analyze molecular properties of PSTG_23616 . The yeast secretion system, agrobacterium-mediated transient expression system, quantitative real-time PCR （qRT-PCR） technique and subeellular localization assays were applied to functional characteriza- tion ofPSTG_23616 . The full-length of genomic DNA of PSTG_23616 was 937 bp,and the open read- ing frame （ORF） of PSTG_236J6 was 699 bp. Sequence analysis indicated that the signal peptide of PSTG_23616 was located in the N terminal 1--22 amino acids. The yeast secretion system assays indi- cated PSTG_23616 was a typical secreted protein with the signal peptide for protein secretion. The se- creted proteinPSTG_23616 could inhibit the PCD triggered by apoptosis protein BAX in mice. qRT- PCR assays revealed that PSTG_23616 transcripts was up-regulated during early infection stages of Pst and the maximum expression reached at 24 h. However,the expression of PSTG_23616 was down- regulated gradually after 72 h till to 264 h. Subcellular localization assays revealed thatPSTG_23616 was localized throughout the cells of wheat protoplasts or tobacco ceils. Our data showed thatPSTG_ 23616 may be involved in differentiation of infection structures of Pst and had a virulence role during Pst-wheat interaction.