小麦条锈菌果胶酶基因PsPL1的克隆与功能分析

Cloning and functional analysis of PsPL1 from Puccinia striiformis f.sp.tritici

【目的】研究小麦条锈菌果胶酶基因PsPL1的功能,确定其在小麦条锈菌侵染过程中的作用,为揭示小麦与病原菌互作的分子机理奠定理论基础。【方法】利用RACE技术扩增PsPL1基因全长,通过qRT-PCR对PsPL1的表达特征进行分析,并在酵母系统中验证其编码蛋白信号肽的分泌功能,最后通过HIGS技术沉默PsPL1基因,确定其在条锈菌侵染小麦过程中的作用。【结果】克隆得到PsPL1基因全长,其ORF长471bp,编码157个氨基酸;经预测PsPL1蛋白包含一个果胶酶保守结构域,N端含有一段由21个氨基酸组成的信号肽序列;经酵母系统验证,确定PsPL1蛋白信号肽具有分泌功能;qRT-PCR分析发现,PsPL1在条锈菌侵染早期表达上调;利用HIGS技术沉默PsPL1基因后,条锈菌的产孢量没有发生明显变化。【结论】成功克隆了小麦条锈菌果胶酶基因PsPL1,明确了其分泌特性及表达特征。

【Objective】This study investigated the role of pectin enzyme gene PsPL1 from Puccinia striiformis f.sp.tritici and determined its function in wheat stripe rust infection to provide theoretical foundation for the molecular mechanism of wheat and pathogen interactions.【Method】RACE and qRTPCR technology were used to amplify PsPL1 length gene and analyze its expression pattern.Then,its secretion system in yeast was verified and HIGS technology was used to silence PsPL1 gene and determine its role in wheat rust infection.【Result】This study obtained the full-length of PsPL1 with ORF of 471 bp encoding 157 amino acids.The PsPL1 protein contained a conserved pectin lyase domain and a signal peptide with 21 amino acids on the N-terminal,which had secretion function.Gene expression analysis suggested that PsPL1 was up-regulated during the early infection.The sporulation quantity had no significant change after knocking down PsPL1 by HIGS.【Conclusion】PsPL1gene has been cloned successfully,and its secretion function and expression characteristics were determined.