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梨果实褐腐病危害果实,可造成果实运输和储藏过程中严重的经济损失。本研究构建了褐腐病菌侵染梨果实愈伤组织离体系统,对梨褐腐病菌侵染其果实愈伤组织不同时期进行细胞学观察分析,基于cDNA-SRAP技术,分析该过程中差异基因表达,以期分离克隆与梨果实抗病反应过程相关的防卫基因。结果表明:与未侵染的梨果实愈伤组织相比,侵染12～60h过程中梨果实褐腐病菌从表面逐渐深入到内部细胞;30个SRAP引物组合共扩增出457条带,其中差异回收条带数为16条,差异比率为3.5%。最终获得5条差异基因表达条带。核酸序列同源性分析表明,其中1条差异基因片段未搜索到任何同源蛋白,2条差异基因片段经序列比对,序列相似度一致,与苹果属的肉桂醇乙酰脱氢酶(CAD)同源性为96%;其他2条差异基因片段分别与DNA结合蛋白(DBP)和寡肽转运蛋白(OPT)基因序列同源,其同源性为85%和78%,因此暂将这3个基因命名为PbCAD、PbDBP和PbOPT。荧光定量PCR结果表明,PbCAD基因在褐腐病菌侵染梨果实愈伤组织12和24h时相对表达量最高,为对照的2.94和2.66倍;PbOPT基因在褐腐病菌侵染梨果实愈伤组织12～36h时相对表达量明显升高,为对照的2.17～2.46倍,而其他时期表达量均与对照接近;PbDBP基因表达量在整个侵染时期均与对照接近。因此我们推测PbCAD和PbOPT基因可能为梨褐腐病菌侵染梨果实愈伤组织响应的相关防卫基因。
Brown fruits of pear fruit are endangered by fruits and can cause serious economic losses during fruit transportation and storage. In this study, the brown rot pathogen infected pear fruit callus tissue system was constructed, the pear brown rot pathogen infected fruit callus at different stages of cytological analysis, based on the cDNA-SRAP technology, analysis of the process of differential gene Expression, in order to isolate cloned and pear fruit disease-resistant reaction-related defense genes. The results showed that compared with non-infected pear fruit callus, pear brown rot pathogen gradually penetrated into the internal cells from the surface during 12-60 hours of infection. A total of 457 bands were amplified by 30 SRAP primer combinations The number of differential recovery strip was 16, the difference was 3.5%. Finally, five differential gene expression bands were obtained. Nucleotide sequence homology analysis indicated that no homologous protein was found in one of the differential gene fragments, and the two differential gene fragments were aligned by sequence alignment. The similarity of the two sequences was similar to that of apple cinnamon acetyl-dehydrogenase (CAD) The homology was 96%. The other two differential genes were homologous to the DNA binding protein (DBP) and oligopeptide transporter (OPT) gene sequences, respectively, with 85% and 78% identity The genes are named PbCAD, PbDBP and PbOPT. Fluorescent quantitative PCR results showed that PbCAD gene had the highest relative expression level of 2.24 and 2.66 times when the brown rot pathogen invaded pear fruit callus at 12 and 24 hours, respectively. PbOPT gene was highly expressed in pear fruit callus 12 ~ 36h, the relative expression level was 2.17 ~ 2.46 times that of the control, while the expression levels in other periods were close to the control; PbDBP gene expression in the entire infection period were close to the control. Therefore, we hypothesized that the PbCAD and PbOPT genes may be the related defense genes of pear brown rot infected pear fruit callus.