基于转录组测序开发的一个小麦叶锈菌特异分子标记

Development of a Specific Molecular Marker for Wheat Leaf Rust Based on Transcriptome Sequencing

小麦叶锈菌毒性变异频繁,新的毒性小种不断出现,往往导致小麦抗叶锈病品种在推广种植几年后的抗性丧失。因此,开发用于田间小麦叶锈病的诊断与预测预报的特异性分子标记具有重要意义。在前期已经完成小麦叶锈菌PHNT转录组测序的基础上,本研究拟在数据库中挑选候选效应蛋白,利用实时荧光定量PCR明确其在叶锈菌侵染小麦后不同时间点的表达模式,根据差异表达分析筛选出候选效应蛋白,再根据编码候选效应蛋白的基因序列设计引物,对小麦叶锈菌PHNT基因组DNA进行PCR扩增,旨在开发小麦叶锈菌特异性分子标记。结果在转录组数据库中筛选出24个候选效应蛋白,qPCR检测明确其中17个具有明显的表达差异。PCR扩增结果表明,基于候选效应蛋白基因PTTG-05290设计的引物在小麦叶锈菌PHNT中获得一条920 bp的条带,且在检测的25种叶锈菌生理小种中均稳定存在,而在小麦条锈菌、菜豆锈菌、枣树锈菌、柳树锈菌、苹果树锈菌及芦苇锈菌6种锈菌中不能获得有效扩增,表明该引物在小麦叶锈菌中具有特异性。qPCR结果表明,PTTG-05290在叶锈菌侵染小麦4 d时达到表达高峰,表达量为0 d的218倍,而0.5 d时便大量检测到该基因。因此,PTTG-05290可作为分子标记用于小麦叶锈菌的田间早期检测及预测预报,对小麦抗叶锈病品种在田间的合理布局具有重要意义。

New virulence races of the wheat leaf rust pathogen emerge continuously,due to frequent mutations in pathogenicity,often resulting in loss of resistance in wheat cultivars originally resistant to leaf rust.So,it is important to develop a specific molecular marker for diagnosis and prediction of wheat leaf rust in the field.On the basis of our previous studies of completing transcriptome sequencing of wheat leaf rust fungus PHNT,in this study we selected candidates of effectors from the wheat leaf rust fungus PHNT transcriptome database,used real-time fluorescence quantitative PCR(qPCR)to detect their expression patterns at different time points after leaf rust fungus infection with wheat,then designed primers according to the sequences of the selected candidates of effectors based on expression differences,conducted PCR amplification using genome DNA of PHNT as templates to screen a specific molecular marker for wheat leaf rust.As a result,a total of 24 candidates of effectors were screened based on the transcriptome database,and 17 expressed significantly difference by qPCR.The PCR results showed that the candidates of effector PTTG-05290 had a specific band of 920bp appearing in the wheat leaf rust fungus PHNT,and the specific band was detected in 25 leaf rust races,but was absent in six rust fungi of wheat stripe rust,bean rust,jujube rust,willow rust,apple rust and reed rust,which indicated that the marker was specific for wheat leaf rust fungus.In addition,the results of qPCR showed that the expression level of PTTG-05290 is 218 times higher at the expression peak(4 dpi)than that at 0 dpi,and the gene could be detected in large quantities at 0.5 dpi.So,the specific molecular marker can be used for the detection and prediction of leaf rust in the field at an early stage,providing a shortcut for the adoption of wheat cultivars of resistance to leaf rust.