Cloning and Analysis of T-DNA Targeted Gene of Colletotrichum gloeosporioides Pathogenicity-Defective Strain Cs16

Abstract

In preliminary stage, the nucleotide sequence of Cs16 Colletotrichum gloeosporioides associated gene obtained from database of (NCBI), analyzed with Clustalx 1.83 in order to assess the deduced amino acid sequences. The full-length sequences of Cs16 gene is including with specific Open Reading Frame (ORF) having 3’ and 5’ UTR was obtained. In order to assess the relationship of C. gloeosporioides associated CS16 gene with other members, a combined rooted neighbor-joining (NJ) tree was generated through MEGA 4.5 by following 1000 bootstrap replicates. The resulted alignment predicts that Cs16 gene showed entirely diverse sequences from other species and has no any conserved domain sequences between them. In order to scrutinize the full length nucleotide sequences, the gene functional cloning of Cs16 gene has been carried out by PCR amplification. To examine the functional analysis of Cs16 gene in C. gloeosporioides and its regulatory mechanisms, the over expressing vector (pGapneoR26) was transformed into Agrobacterium strain LBA4404 that resulted successful detection of GFP-Cs16 expression into C. gloeosporioides transformants and it was also substantially detected by fluorescence microscopy. The T-DNA intentional mutants of C. gloeosporioides mycelia illustrated phonotypic different intensification, colour, colony growth rate than wild type isolated C. gloeosporioides STIJ6 strain.