对甘蔗受黑穗病菌侵染后差异表达基因的分离与鉴定

采用12个3''锚锭引物和8个5''随机引物构建引物组合, 运用DDRT-PCR差异显示方法, 分析NCo376和F134两品种接种黑穗病菌前后的基因表达差异。经克隆、测序和半定量RT-PCR验证, 获得7条真实差异表达片段, 这些片段分别同GenBank中编码细胞色素C氧化酶基因、核糖体蛋白基因、NAD-依赖型苹果酸脱氢酶基因、氨基转移酶基因、乙烯响应相关结合蛋白基因、RNA聚合酶特异转录起始因子和反转录转座子的序列的同源性达28%~99%。半定量RT-PCR分析表明, 细胞色素C氧化酶基因的表达受甘蔗黑穗病菌和水杨酸的调控, 不依赖于过氧化氢的作用机制, 在甘蔗根、茎、叶中表达的抗病基因组成型表达, 但表达水平较低。推测甘蔗受黑穗病菌侵染后, 可能通过细胞色素C氧化酶基因的诱导, 促使植保素合成增多, 以此抵抗或抑制病原菌的胁迫。该结果丰富了甘蔗与黑穗病菌互作的分子机制研究, 为甘蔗抗黑穗病分子育种奠定了基础。

Isolation and Identification of Differentially Expressed Genes in Sugar-cane Infected by Ustilago scitaminea

Sugarcanesmut, caused by Ustilago scitaminea Syd., is a most severe fungal disease resulting in heavy economic losses in sugarcane production. The best way to control the disease is to breed resistant varieties. The objective of this study was to survey the molecular mechanism of sugarcane smut resistance. Twosugarcane varieties infected by smut, consisting of NCo376 with high resistance and F134 with susceptibility, were detected to reveal the differential expression of genes regulating resistance to smut with twelve anchored primers and eight random primers via DDRT-PCR. Seven differentially expressed polymorphic fregments were obtained by coloning, sequencing and semi-quantitative RT-PCR validation. The results of Blast in GenBank showed that they shared high homology (52%–97%) with cytochrome C oxidase (CCO) gene, ribosomal protein gene, NAD-dependent malic enzyme gene, aminotransferase gene, binding protein gene and retrotransposon, respectively. The results of semi-quantitative RT-PCR indicated that while CCO gene expression was regulated by U.sticaminea and salicylic acid (SA), its expression was independent on H₂O_2. Besides, CCO gene expressed in root, stalk and leaf of sugarcane at a relatively low level. From described above, it was inferred that the synthesisof phytoalexin induced by CCO inhibited the pathogen after infection. The results provided useful information for further understanding the molecular mechanism of sugarcane-smut interaction.