细菌性条斑病菌JH01诱导水稻抗病均一化差减文库的构建

目的]构建细菌性条斑病菌诱导下的水稻抗病均一化差减c DNA文库,对获得的差异表达ESTs进行生物信息学及抗病相关基因的表达分析。方法]以水稻IR26(Tester)和两优培九(Driver)幼苗5~6叶期叶片为材料,采用双链特异性核酸酶(DSN)介导的均一化消减杂交技术,构建细菌性条斑病菌JH01诱导的水稻抗病相关基因差减c DNA文库,以p LB-F和p LB-R为引物,通过菌液PCR扩增对差减文库的质量进行检测。结果]c DNA文库的插入片段分布在0.5~2.0 kb,平均大小约为1.0 kb,重组率达95%。序列测定分析发现,随机挑取的504个克隆中有98条差异表达基因;经BLAST比对和GO注释发现,59条序列具有同源基因,分别参与信号转导、能量代谢、过敏性坏死反应、防卫反应等;另外39条序列无相似基因,有待进一步研究。通过实时荧光定量PCR发现,从该文库中分离得到的Os NDPK4参与细菌性条斑病菌JH01诱导的水稻防卫反应。结论]水稻受细菌性条斑病菌侵染的c DNA文库质量较好,为研究条斑病菌致病性因子与水稻互作机制奠定基础。

Construction of a Normalized Subtractive cDNA Library of Rice Inoculated with Xanthomonas oryzae pv.oryzacola JH01

Objective] A normalized subtractive cDNA library of rice inoculated with Xanthomonas oryzae pv.oryzacola JH01 was constructed,the expression analysis of biological information and disease resistance related genes was conducted on obtained differential expression of ESTs.Method] In this study,using seedling leaves of rice ‘IR26’ (Tester) and ‘ Liangyoupeijiu’ (Driver) as experimental materials,a subtracted cDNA library of rice inoculated by Xanthomonas oryzae JH01was constructed by the method based on subtractive hybridization of the DSN (duplex-specific nuclease) mediated subtractive hybridization.The quality of the subtractive library was detected by PCR amplification using primers pLB-F and pLB-R.Result] The results demonstrated that the sizes of inserted fragments in the vector were between 0.5-2.0 kb,that the average size was about 1 kb,and that 95％ of the library clones were recombinants.504 clones were randomly selected,and 98 genes were obtained after clearing pollution,redundant,low quality sequences,and 59 genes were found to have homologous genes in analysis by BLAST.Gene ontology showed that 59 genes were related with resistance signals transduction,elementary metabolic,hypersensitive response,defense response et al.The other 39 sequences had no similar genes,which need further study.By real-time fluorescent quantitative PCR,OsNDPK4 isolated from the library was found to be involved in defense responses in rice induced by Xanthomonas oryzae JH01.Conclusion] The above suggests that the constructed cDNA library of rice is of good quality and of use for isolation of interacting proteins.