条锈菌诱导的小麦抗病与感病近等基因系SSH文库构建及分析

为分析条锈菌诱导下的小麦抗病与感病近等基因系之间差异表达的基因,以接种小麦条锈菌CY26小种的抗病近等基因系Yr4/6×Taichung 29幼苗叶片cDNA作为实验方,接种CY26的感病亲本Taichung 29幼苗叶片cDNA为驱动方,利用抑制消减杂交(SSH)技术构建了一个包含1 300余克隆的消减文库。对文库中600个克隆进行了反向Northern点杂交筛选,对获得的阳性克隆进一步进行了Northern杂交验证,获得显著差异的克隆12个。经测序和BlastX分析,其中6个差异表达序列的推测产物分别为亮氨酸重复序列蛋白、过氧化氢酶、硫氧还蛋白、RNA结合蛋白、抗坏血酸过氧化物酶和热激蛋白。除亮氨酸重复序列为信号传导类蛋白外、其他几个均为抗病防御类蛋白。

Construction and analysis of the SSH library with the resistant wheat near-isogenic line and its susceptible parent infected by Puccinia striiformis Westend. f. sp. tritici

To analyze the differentially expressed genes between resistant and susceptible wheat near-isogenic lines infected by Puccinia striiformis Westend. f. sp. tritici, a subtractive library containing about 1300 clones was constructed using suppression subtractive hybridization (SSH) in which the cDNA from resistant Yr4/6 × Taichung 29 seedlings inoculated with race CY26 was used as the tester, and the corresponding cDNA from susceptible Taichung 29 as the driver. Six hundred clones from the library were analyzed with reverse Northern blot. The positive clones were further tested by Northern blotting analysis. Twelve clones were verified and showed significant difference. By means of sequencing and BlastX analysis, six function-known differentially expressed sequences were detected, and their putative products were leucine-rich repeat protein, catalase, thioredoxin H-type, RNA binding protein, ascorbate peroxidase, and heat shock protein, respectively. Among them, leucine-rich repeat protein belongs to signal transduction protein, and others belong to defense response protein.