Fusarium oxysporum genes expressed in planta

1)尖孢镰刀菌Fusarium oxporum f.sp.对黄瓜木质部汁液中特异表达的黄瓜果胶(Foc)基因在mRNA和蛋白质水平进行了检测。对用抑制性消减杂交方法分离的29个Foc基因克隆进行了测序，并进行了同源性搜索。进一步检测了两个与NIPSNAP基因和可能的阳离子转运蛋白基因同源性较高的克隆在植物中的表达。根据这两个序列设计的特异引物进行RT-PCR分析表明，它们对应的基因实际上在寄主植物中表达。用双向SDS-聚丙烯酰胺凝胶电泳法分析了黄瓜木质部汁液中积累的FOC蛋白。从凝胶中提取了22个蛋白质点，进行了多肽质量指纹图谱分析。序列比对搜索表明，数据库中没有与获得的多肽质量指纹图谱匹配的蛋白质。2)尖孢镰刀菌番茄红素酶基因(Fotoml)。已知在植物中表达的番茄被选为基因干扰的靶基因。获得的18个突变体对α-番茄红素的敏感性增加，番茄红素酶活性降低。接种试验表明，突变株的致病力降低，提示番茄红素酶在该真菌致病中起重要作用。Norome印迹分析表明，当其中一个突变株被α-Tomatine诱导时，Fotoml的反义RNA表达，这表明至少在该突变株中，转录后基因沉默参与了对番茄红素酶活性的抑制。

1) Fusarium oxysporum f. sp. cucumerinum (Foc) genes expressed specifically in cucumber xylem sap were examined at mRNA and protein levels. Twenty-nine Foc cDNA clones isolated by suppression subtractive hybridization were sequenced and the resultant nucleotide sequences were subjected to homology searches. Two cDNA clones showing high homology with NIPSNAP gene and putative cation transporter gene were further examined for their expression in planta. RT-PCR analysis with specific primers designed from the two sequences revealed that their corresponding genes are actually expressed in host plants. Foc proteins accumulated in cucumber xylem sap in vitro were analyzed by two-dimensional SDS polyacrylamide gel electrophoresis. Twenty-two protein spots were extracted from the gels and subjected to peptide mass fingerprinting analysis. Sequence alignment searches showed that no proteins in databases were matched to the peptide mass fingerprintings obtained.2) Tomatinase gene (Fotoml) of Fusarium oxysporum f. sp. lycopersici which has been known to be expressed in planta was chosen as a target gene for gene disruption. Eighteen mutants obtained showed increase in sensitivity to α-tomatine and decrease in tomatinase activity. Inoculation assay showed reduced pathogenicity of the mutants, suggesting an important role of tomatinase in pathogenicity of this fungus. Northem blot analysis showed that antisense RNA of Fotoml was expressed when one of the mutants was induced by α-tomatine, suggesting posttranscreptional gene silencing is involved in the suppression of tomatinase activity at least in this mutant strain.