第二代测序技术用于水稻和稻瘟菌互作早期转录组的分析

摘要

稻瘟菌为了实现对水稻的有效侵染,在侵染水稻时可能通过表达和转运一定数量的效应蛋白进入到水稻细胞,抑制和干扰水稻的先天免疫机制.文章利用Solexa第二代测序技术,通过开展水稻和稻瘟菌互作早期转录组的测定和分析,克隆和鉴定在互作早期表达的稻瘟菌效应蛋白基因.利用序列同源比对,我们从总计约12.5 M条序列标签中,分离和鉴定了338 942条来源于稻瘟菌的序列,并最终定位到779个稻瘟菌预测基因.其中108个基因很可能参与了水稻和稻瘟菌互作过程,42个基因为预测的分泌蛋白基因.通过RT-PCR分析,最终确认了42个预测分泌蛋白基因中有12个基因在侵染水稻早期有显著的表达,而其中有4个基因表现为侵染早期特异表达.文章尝试利用第二代测序技术实现稻瘟菌侵染早期特异表达基因,尤其是分泌蛋白基因的快速克隆和鉴定,为稻瘟菌效应蛋白基因的克隆和功能鉴定提供了较为有意义的探索.%Magnaporthe oryzae is speculated to express and translocate a set of effector proteins to interfere with rice innate immunity and to promote its invasion. In this study, we carried out a transcriptome analysis of early interaction be-tween rice and M. Oryzae using a next generation sequencing technology to identify putative effector protein genes. We totally obtained 338 942 M. Oryzae derived Sequence tags from around 12.5 M gross mixed tags by refering the genome sequence of reference strain 70-15. These M. Oryzae sequences were finally mapped to 779 predicted genes. Of these 799 genes, 108 were predicted to be involved in the interaction between rice and M. Oryzae and 42 encoded proteins predicted to be secreted. Expression of these 42 putative secreted protein genes were further assessed using a reverse transcription po-lymerase chain reaction (RT-PCR) method, which revealed that 12 of them were expressed during the infection. Four of these 12 expressed genes were further confirmed to be expressed specifically during the infection. This study demonstrated an attempt to isolate M. Oryzae genes, especially those putative effector protein genes, which are expressed specifically at the early interaction stage using a next-generation sequencing technology.