Function Analysis Of Laccase Genes (StLAC) In Melanin Biosynthesis Pathway Of Setosphaeria Turcica

This research intend to determine the laccases'function of melanin synthesis and pathogenicity in Setosphaeria turcica. It had been found that laccase could accelerate the extention of S. turcica against the host tissue. A null StLAC1 mutant was obtained using homologous recombination and it was found that the gene played a critical role in the melanin synthesis. Laccase gene StLAC2 of S. turcica was cloned by using PCR and RACE technologies, The amino acid sequence of StLAC2 gene shared 29.22% homology with Stlac1 at the protein level. Main results in this paper were as follows:1. Laccase activity was compared between S. turcica and nine other plant pathogenic fungi which played critical roles in lignin degradation. S. turcica contained the higher intracellular laccase activity, which was 18.984 U·mL^-1. The analysis of histopathology in S. turcica showed that corn leaves were oxidated by laccase which could promote the expansion of pathogen against the host.2. By using homologous recombination technology, StLAC1 gene-knockout vector was transformed into S. turcica by PEG-mediated transformation method. A StLAC1 gene-deletion transformants were screened by hygromycin B. One StLAC1 gene-disruption mutant named asâ–³StLAC1 was determined by PCR, Southern blot and semiquantitative RT-PCR analysis.3. The mutantâ–³StLAC1 displayed a gray-white colony which had less melanization compare with the wide type. Unlike the wide type, the null StLAC1 mutant could not produce conidia, it was transparent and had irregular-shaped mycelium, had slower vegetative growth rate, enhanced osmotic ability, weaked turgor pressure and penetration of appressorium, decreased laccase activity. StLAC1 gene involved in melanin biosynthesis and related to penetration of appressorium, form of conidiophore, respectively.4. StLAC2 was cloned using PCR and RACE technologies. StLAC2 possess 1 763 bp DNA sequence which contained 1 665 bp coding region, encoded 554 amino acids and consisted of 3 exons and 2 introns. The amino acid sequence of StLAC1 gene shared 45%⁷6% indentity with other fungal laccases, shared 29.22% with the amino acid sequence of StLAC1 gene at the protein level. Analyzed the cluster tree and conserved domains, we found that Stlac1 and Stlac2 had the conserved copper binding domains, but belonged to different branches of phylogenetic trees and related distantly. Southern blot results showed that StLAC2 gene had single copy in the genome of S. turcica.