Coloning And Functional Identification Of Laccase Gene(Lac1) In Pathogenicity From Colletotrichum Gloeosporioides-the Pathogen Of Mango Anthracnose Disease

Mango (Mangifera indica L.) was one of the five famous tropical fruit in the world. Mango anthracnose was one of the most serious diseases that influenced mango industry and occurs at all mango region. Colletotrichum gloeosporioides was the main pathogens of mango anthracnose. Currently, domestic and foreign studies mainly focused on pathogen biology and disease epidemiology as well as control. However, there has been little research on pathogen molecular biology, especially in pathogen-related genes and their regulations. Resaerch on the pathogeneisis-related genes in Mango-C. gloeosporioides system would be of great academic value in terms of revealing the pathogenesis and understanding the interaction mechanism between C. gloeosporioides and its hosts. Furthermore, such studies may be useful for finding new target sites to improve control of mango anthracnose.In this study, full-length sequences of a laccase gene Lacl was obtained using homology-based clone from the genome of C. gloeosporioides from mango. The disrupted genes were designed by insertion mutation with the Green Fluorescent Protein gene-hygromycin B phosphotransferase gene (gfp::hygB) and gene knockout mutants were obtained by polyethylene glycol-mediated transformation of fungal protoplasts, and the phenotypes of mutant were also tested. The results were mainly as follows:1. C. gloeosporioides strain A2was considered as high capacity of producing laccase by agar selecting method using guaiacol as substrates.Laccase could accelerate the extention of C. gloeosporioides in the mango tissue. A laccase gene was cloned from C. gloeosporioides on mango by SEFA-PCR and RT-PCR, the results showed that the complete DNA and cDNA sequence of Lacl gene were2996bp and1768bp in size, respectively, with three introns of47bp,50bp and64bp, respectively (being within the583-629,785-834and977-1040bases),encoding a putative protein of590-amino acid with a molecular weight of65.46kDa and isoelectric point of5.36, and the ahead twenty-two amino acid was a putative signal peptide sequence. Phylogenetic analysis indicated that putative protein of Lacl was homologous to C. lagenarium laccase (BAB32575.1) with a similarity of73%, and was also homologous to copper-containing oxidases in some other fungi at some similarities. Therefore, the Lacl gene from C. gloeosporioides in this study was an laccase gene. The sequence had been submitted to GenBank (JQ762259.1).2. The semi-quantitative RT-PCR results showed that in different infection stages, the Lacl expression was different with the lowest at6h and the highest at9h after inoculation. Using the In-FusionR HD Cloning Kit, the Lacl gene knockout vector pA2LFGHB was constructed. A recombined Lacl gene fragment from the knockout vector containing the gfp::whygB gene was amplified, and used to transform C. gloeosporioides protoplast. After PDA plate screening, PCR verification, and Southern blot hybridization, a laccase gene Lacl knockout mutant was confirmed, and named,△A2LAC1. Compared with the wild-type strain, mutant AA2LAC1displayed a close to white colony from grey-black, no appressoria formation on the tip of germ tube and incapable of secreting laccase. Sporulation quantity, activity of cell wall degrading enzymes (CWDEs) and content of intracellular melanin decreased significantly. Aerial mycelial mass with less branches and mycelial growth rate significantly reduced. The mycelium was more sensitive to high temperature, whereas the optimal range of pH widen and osmotic stress response to a variety of salts and antioxidant ability enhanced. The utilization ability for part carbon and nitrogen sources was different from the wild-type. The results of pathogenicity test shown that smaller necrotic lesions were observed on detached wound leaves and ripe mango peel inoculated with mycelia plugs of mutant AA2LAC1, while were not on unwounded leaves and ripe mango peel. The above findings demonstrated that the laccase Lacl might be play a vital regulatory role in mycelial growth, development, cell differentiation, and pigmentation, as well as conidium formation, laccaes and CWDEs production, adaptability to the environment, and pathogenicity of C. gloeosporioides on mango.