Abstract: 【Objective】 The objective of this study is to analyze the biological phenotypes of the pathogenicity-defective mutant strain B-1015, to clone its T-DNA integration flanking sequences, and finally to reveal the functions of targeted genes in pathogenesis of Ustilaginoidea virens. The study will contribute to better understanding of the molecular pathogenesis of U. virens. 【Method】 Biological phenotypes of B-1015 were analyzed by testing growth rate, sporulation ability, spore germination rate and pathogenicity. The copy numbers of T-DNA inserted into B-1015 were identified by Southern blot. The flanking sequences of T-DNA were cloned by hiTail-PCR and the whole gene sequences of the T-DNA targeted genes were cloned by RACE PCR. The genes expression were detected by RT-PCR. 【Result】By comparing with wild type strain P1, the sporulation ability and growth rate of the B-1015 were significantly declined, but the spore germination rate had no significant difference. The inserted mutant lost its pathogenicity in rice. Molecular detection analysis confirmed that the B-1015 was a stable single T-DNA insertional mutant. The T-DNA flanking sequences were obtained by hiTail-PCR. Two genes, Uvt-1015R and Uvt-1015L, were cloned by RACE PCR. Gene sequence analysis showed that Uvt-1015R contained a 948 bp open reading frame, which could encode a 295 amino acid protein, a 341 bp 5′-UTR and a 271 bp 3′-UTR. Another gene, Uvt-1015L, comprised a 351 bp open reading frame coding a 116 amino acid protein, a 31 bp 5′-UTR and a 174 bp 3′-UTR. By searching in the known protein, the Uvt-1015R and Uvt-1015L genes showed no homology of any protein. Sequence alignment showed that the flanking sequences of T-DNA were non-adjacent in the wild type genome and the T-DNA disrupted the two genes sequence. RT-PCR analysis confirmed that compared to P1 strain, the transcripts of Uvt-1015R was significantly decreased, and the transcripts of Uvt-1015L was not detected any more. 【Conclusion】 In B-1015, both the T-DNA insertion and chromosomal rearrangement caused important biological phenotype mutant, thus leading to the loss of pathogenicity.

Key words: Ustilaginoidea virens , pathogenicity , T-DNA , gene clone