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以抗黑星病黄瓜材料HX1为试材,接种黑星病菌(Cladosporium cucumerinum)2 h、8 h、20 h、32 h和72 h的叶片作为试验方(Tester),相应的未接种叶片作为对照方(Driver),利用SSH技术,构建了黑星病菌侵染初期的正向和反向cDNA-SSH文库。用巢式引物PCR检测插入片段,获得了200个阳性克隆,通过测序,除去重复序列,共得到105个Unique ESTs,其中50个为singleton,55个为contings。与非冗余蛋白数据库进行BLASTx比对,结果显示,17条ESTs未找到同源序列,88条非重复序列和已知基因的同源性较高,占全部ESTs序列的83.8%,其中86条ESTs与非冗余蛋白数据库已知功能的蛋白具有高度的相似性。结合高密度点阵膜杂交差异筛选,阳性率为75.0%。经初步分析这些序列的功能,差异表达的ESTs功能涉及能量和基础代谢、信号转导、蛋白和核酸代谢、光合作用及逆境中特异表达的基因等方面,为研究黄瓜抗黑星病基因提供了依据。
The anti-Sci-Cucumber material HX1 was used as test material to inoculate the leaves of Cladosporium cucumerinum for 2 h, 8 h, 20 h, 32 h and 72 h, respectively. The uninoculated leaves were used as control Driver, utilizing SSH technology, constructed the forward and reverse cDNA-SSH libraries of the initial stage of infection of Scabs. A total of 105 unique ESTs were obtained by PCR using nested primer PCR and 200 positive clones. A total of 105 unique ESTs were obtained, of which 50 were singleton and 55 were contings. BLASTx alignment with non-redundant protein database showed that no homologous sequences were found in 17 ESTs, 88 non-repetitive sequences shared high homology with known genes, accounting for 83.8% of all ESTs, of which 86 ESTs have a high degree of similarity to known proteins in non-redundant protein databases. Combined with high-density dot matrix hybridization differential screening, the positive rate was 75.0%. After preliminary analysis of the function of these sequences, the function of differentially expressed ESTs involved in energy and basal metabolism, signal transduction, protein and nucleic acid metabolism, photosynthesis and genes specifically expressed in adversity, etc. provided a in accordance with.