梨果实愈伤组织褐腐病菌侵染过程cDNA-SRAP差异分析

Analysis of Differential Gene Expression Induced in Pear Fruit Callus by Monilinia fructicola Infection Using cDNA-SRAP

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。

Moniliniafructicola causes serious loss in pear fruit during transportation and storage. In order to clone defence-related genes during infection process, in vitro systems based on pear fruit callus infected by M. fructicola were constructed and gene expression levels were characterized by cDNA-SRAP （cDNA sequence- related amplified polymorphism） at different time point （control, 12, 24, 36, 48 and 60 h）. We observed that 457 fragments were amplified from 30 SRAP primer groups, out of which 16 fragments （3.5% of the total） were dif- ferentially expressed in polyacrylamide gel electrophoresis （PAGE） detection. Those 16 fragments were re-amplified for strip recovery by agarose gel electrophoresis. Only five were successfully recovered and subsequently sequenced. Sequence homology analysis of those fragments by Blastn showed that： one had no homology to any known sequences in the NCBI data base; two of them are of identical sequence; the three different sequences were of high similarity to cinnamyl alcohol dehydrogenase （CAD）, DNA binding-protein （DBP） and oligo-peptide transporter protein （OPT）, with the identity being about 96%, 85% and 78%, respectively. They were hence named as PbDBP,, PbOPT and PbCAT. Differential expression patterns of PbDBP, PbOPT and PbCAT were observed at different time points （0-60 h） using qRT-PCR. PbCAD expression levels in callus co-cultured with M. fructicola for 12 and 24 hours were 2.94- and 2.66-fold higher than the control, respectively. The high- er expression levels of PbOPT gene was observed after co-cultured with M. fructicola for 12-36 h, they were 2.46-, 2.17-, and 2.34-fold higher than the control, respectively. However, there were no significant higher ex- pression levels detected for PbDBP at all infected period. These results suggested that PbCAD and PbOPT could be defence-related genes during the process of brown rot infection in pear fruit callus.