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采用cDNA-AFLP(complementary DNA-amplified fragment length polymorphism)技术,以高抗疫霉病辣椒品种‘L11’接种疫霉菌的叶片为材料,对6叶期辣椒幼苗灌根接种疫霉菌侵染后6个时间点的叶片基因表达谱进行了差异分析。利用86对引物组合,共筛选出了363条差异表达的cDNA片段,对其中100个表达差异显著的片段进行测序,最终得到73个差异片段的核苷酸序列。经Blastx比对和功能分类分析,其中45个序列与已报道的功能基因具较高同源性,其功能涉及蛋白质代谢、信号转导、防御反应、碳水化合物代谢和光合作用等。在差异片段序列基础上设计特异引物,采用基于PCR技术的96孔文库筛选方法,从文库中分离得到2个差异基因的全长cDNA,荧光定量PCR分析结果显示其表达模式符合cDNA-AFLP表达谱。本研究利用c DNA-AFLP技术筛选到的一批与抗疫霉病相关的基因片段,可进一步用于功能鉴定和分子育种。
By using cDNA-AFLP (complementary DNA-amplified fragment length polymorphism) technique, the leaves of 6-leaf-capped pepper seedlings were inoculated with Phytophthora infestans after inoculation with Phytophthora capsici Differences in leaf gene expression profiles at the time point were analyzed. A total of 363 differentially expressed cDNA fragments were screened out using 86 pairs of primer combinations, and 100 of them were sequenced. The nucleotide sequences of 73 differentially expressed fragments were obtained. Among them, 45 sequences have high homology with the reported functional genes, and their functions are involved in protein metabolism, signal transduction, defense response, carbohydrate metabolism and photosynthesis. The specific primers were designed based on the differential fragment sequences. The full-length cDNA of two differential genes was isolated from the library by PCR-based 96-well library screening method. The results of quantitative PCR showed that the expression pattern was in accordance with the cDNA-AFLP expression profile . In this study, a number of genes related to Phytophthora infestans were screened by c DNA-AFLP technology and could be further used for functional identification and molecular breeding.