The analysis of T-DNA insertional Colletotrichum gloeosporioides in Stylo pathogenicity-weakened mutant strain 1681

摘要

Biological phenotypes of mutant strain 1681 that was the pathogenicity-reduced strain in Colletotrichum gloeosporioides of Stylosanthes guianensia were analyzed by testing growth rate, colonial morphologies and sporulation ability. TAIL-PCR was used to identify the T-DNA integration site and the genes of flanking right site of the T-DNA. The sequences was analyzed by local blast and the gene and the function were predicted by bioinformatics methods. The results showed that the pathogenicity of the mutant 1681 was reduced by comparing with wild type strain CH008 in Colletotrichum gloeosporioides of Stylosanthes guianensis. The growth rate between strain 1681(1.42±0.02)cm/d and CH008(1.41±0.03)cm/d had no significant difference. The sporulation between strain 1681(1.50±0.25)×10~6/mL and CH008(9.26±0.14)×10~6/mL had significant difference. The spore germination rate between strain 1681(0.00±0.00)% and CH008(91.14±6.66)% had significant difference. In contrast the colony diameter and colony morphology had no significant difference. Two sequences were cloned by TAIL-PCR. The length of the RB flanking sequence was 412 bp and the LB flanking sequence was 381 bp. The blast result showed that the sequence had 100% homology to genome of wild type strain CH008 in Colletotrichum gloeosporioides of Stylosanthes guianensis. The bioinformatics analysis showed the T-DNA maker the area of initial exon. It contains a complete open reading frame (ORF), encoding 682 amino acid. By predicting the function of sequence in NCBI, the blast result showed that the sequence had 99% homology to gene Nara gc5 phenylalanyl-tRNA synthetase (PheRS) beta chain core domain. It belongs to the class II aminoacyl-tRNA synthetases (aaRS) like-core super family based upon its structure.