月季响应黑斑病的早期差异表达基因分析

The Analysis of Differential Expression Genes for Rose Early Responding to Black-spot Disease

为了获得月季抗黑斑病的相关基因,探究月季抗病分子机制,以月季高抗黑斑病品种‘粉和平’叶片为材料,采用单孢子离体接种法对其接种黑斑病菌,以接种24、48和72 h的c DNA等量混合样为检测子,以相应对照的c DNA混合样为驱动子,构建抑制差减杂交文库。挑选200个阳性克隆进行测序、BLASTx比对分析,获得37条非冗余EST序列,其中32条序列在蛋白数据库中具有同源序列。将获得的EST序列导入相应样本的转录组测序数据库,以其在两个样本中的RPKM比值作为基因相对表达量。结果表明,接种病原菌后,上调表达基因涉及信号识别、离子转运、活性氧清除、光呼吸途径及苯丙烷代谢等方面,其中以活性氧清除、光呼吸途径和苯丙烷代谢途径的基因数最多。这一结果说明月季抗病是通过多基因的协同作用实现的,活性氧清除、光呼吸途径和苯丙烷代谢途径在月季抵抗黑斑病菌过程中发挥重要作用。

Black-spot disease is the most serious disease for roses. In order to isolate the genes related with the rose resistance and further to explore the interaction molecule mechanism between rose and Diplocarpon rosae Wolf.‘Pink Peace'as the high resistance variety in the preliminary screening was selected as the material,the c DNA of its inoculated leaves in 24 h,48 h and 72 h were mixed equivalently,and the mixture was regarded as the tester,the c DNA mixture of non-inoculated leaves corresponding hours were used as the driver. Based on these,the subtracted c DNA library were builted. A total of 200 positive clones were picked and sequenced,and 37 non-redundant sequences were obtained. Among them,32 ESTs showed similarity to known sequences in Gen Bank. Then,all 37 EST sequences were imported to the database of transcriptome sequencing built with the samples of the same two mixtures,and their RPKM ratios of the inoculated samples to non-inoculated ones were regarded as the gene relative expression. The results showed that the genes including ion transport,reactive oxygen removal,photorespiration and phenylpropanoid pathway and so on were up-regulated in the inoculated samples,and the genes involved in photorespiration,reactive oxygen removal and phenylpropanoid pathway were more,which suggested that multiple genes involved in the rose defending system. Among of them,reactive oxygen removal,photorespiration and phenylpropanoid pathway are playing important roles in rose's resistance to black spot pathogen.