Isolation Of The Flanking Gene Of T-DNA Insertional Ustilaginoidea Virens Mutants B-726 And B-1015

In this study, the T-DNA insertional pathogenicity deficiency mutant strain B-726 and pathogenicity-defective mutant strain B-1015 were screened through pathogenic analysis from T-DNA insertional Ustilaginoidea virens mutant library. Phenotypic analysis of mutant strain B-726 showed that, compared with the wild-type strain P1, the growth rate of B-726 were declined on MM?TB3 and PSA plate. The sporulation ability of B-726 were declined significantly in PS liquid medium. Using the genomic of subcultured five generations B-726 as a template, PCR analysis the stability of the T-DNA insert. The result showed that T-DNA could be stably inherited. Genomic Southern bolt analysis confirmed that B-726 was a single T-DNA insertional event. The flanking sequence of T-DNA was cloned using TAIL-PCR and the completed gene sequence in the flanking was cloned by RACE-PCR. The total length of the gene was 1296bp, the gene contains two exons and a length of 88 bp of intron, the exons were 406 bp and 798 bp, respectively. The complete cDNA sequence open reading frame analysis showed that the gene comprised a 465 bp open reading frame, which could be encoded a 154 amino acid protein, a 110 bp 5'-UTR and a 631 bp 3'-UTR, the gene named Uvt-726. Sequence analysis showed that the T-DNA insertion site was in the 344 bp upstream of the gene. RT-PCR analysis confirmed that Uvt-726 transcriptions were expressed in B-726 significantly decreased compared to P1. In the known functional protein genes, we did not find any homologous genes. A novel gene maybe associated with pathogenicity of U. virens was cloned, and these results imply that the Uvt-726 might play an important role during the pathogenic process of U. virens.Further analysis the phenotypes of pathogenicity-defective mutant strain B-1015, we found that, compared with the wild type strain P1, the growth rate of B-1015 was declined. The sporulation ability of B-1015 was declined significantly in PS liquid medium. Upload the both ends of T-DNA flanking sequences to U. virens whole genome sequencing database, sequence analysis showed T-DNA insertion may result in U. virens genome rearrangements occurred. Two genes, Uvt1015R and Uvt1015L, were cloned by using RACE PCR. Uvt1015R gene is 1560 bp full-length, contained two exons and a length of 60 bp of intron, the exons were 631 bp and 869 bp, respectively. ORF analysis showed that Uvt1015R comprised a 948 bp open reading frame, which could be encoded a 295 amino acid protein, a 341 bp 5'-UTR and a 271 bp 3'-UTR. The total length of another gene, Uvt1015L, was 556 bp, and the gene did not have intron. Uvt1015L comprised a 351 bp open reading frame coding a 116 amino acid protein, a 31 bp 5'-UTR and a 171 bp 3'-UTR. Sequence analysis revealed that T-DNA insertion in the middle of the both gene sequences, RT-PCR analysis confirmed that in B-1015 Uvt1015R transcriptions were expressed significantly decreased compared to P1, and Uvt1015L was not expression any more. In the known functional protein genes, we did not find any homologous genes. In B-1015, both the T-DNA insertion and chromosomal rearrangement caused important biological phenotype mutant, thus leading to the loss of pathogenicity.Molecular cloning of T-DNA targeted functional genes was the basis of U. virens pathogenesis research, clearly the role of genes in U. virens pathogenic process required for gene functional verification. We constructed UVT-726 gene knockout cassette and streated the hygromycin phosphotransferase (HPH) gene as a selectable marker. Agrobacterium plasmid PC-P1 tk as a vector carrying the knockout cassette ransformated into U. virens by using of Agrobacterium-mediated. The Knockout cassette carried by the T-DNA was transformed into the U. virens, with the help of virulence proteins, T-DNA integrated into the genome of U. virens, or occurred homologous recombination with the U. virens genome, achieved UVT-726 knockout. Transformants were selected with hygromycin resistance. The results were detected by PCR. The objective of this study is to obtain a UVT-726 gene Knockout mutant strain, and analysis of the role of this gene in the pathogenic molecular mechanism of U. virens.