转DsRed荧光蛋白的新月弯孢Curvularia lunata菌株构建

Construction of DsRed-labeling Curvularia lunata

本研究通过农杆菌介导转化方法(ATMT),利用DsRed荧光蛋白基因对玉米弯孢叶斑病致病菌新月弯孢进行遗传转化。通过转化子的荧光蛋白基因和潮霉素B抗性基因的PCR检测,菌丝体和分生孢子的荧光观察,hyg基因的Southern杂交验证,以及荧光蛋白基因插入位点的TAIL-PCR分析,确定了DsRed荧光蛋白基因插入与表达对新月弯孢转化子的影响。结果表明:测定的4株转化子基因组中均成功整合了DsRed荧光蛋白目的基因片段;转化子在生长发育和致病性方面与野生型菌株存在一定差异,分别有2株在产孢量方面略高于野生型菌株,4株转化子在纤维素酶活性和粗毒素致病力方面均低于野生型菌株,有3株转化子在果胶酶活性上较野生型菌株有提高,1株转化子的致病力显著低于野生型菌株;获得其中3个转化子插入位点的侧翼序列。

Red fluorescent protein （DsRed） is a powerful marker used to analyze infection process of pathogenic fungi in plants. In this study we tried to use the ATMT method to transmit DsRed gene into Curvularia lunata, the causal agent of leaf spot in maize. Transformants were identified by PCR analysis of DsRed and hyg genes, observation of red fluorescence in hyphae and spores, southern blotting for hyg gene, and targeting the insertion locus of DsRed gene by TAIL-PCR. The results showed that DsRed gene was successfully integrated into the genome of C. lunata, and caused little change in pathogenicity in DsRed-transgenic strains. Compared with the wild type strain, two transformants were higher in number of conidia; all transformants were lower in cellulase activity and pathogenicity of crude toxin; three transformants were higher in pectinase activity; Cl-DsRed 3 was significantly weakened in pathogenicity. The flanking sequences of T-DNA insertion locus of three transformants were sequensed by hiTAIL-PCR. The transformant, Cl-DsRed 4, was useful in analyzing infection and systematic colo-nization of C. lunata in maize.