关键词: 橡胶树白粉病菌, OhPbs2, 基因克隆, 渗透压, 致病性

Abstract: 【Objective】Pbs2 is one of the important members of HOG-MAPK pathway in MAPK signaling pathway, and plays an important role in osmotic regulation of plant pathogens. Oidium heveae is an obligate parasite. In this paper, the function and effect of Pbs2 of O. heveae were studied by using Colletotrichum gloeosporioides.【Method】The homologous cloning method was used to amplify the OhPbs2 by using the genomic DNA and cDNA as template. The domain of this gene was analyzed by bioinformatics. Phylogenetic analysis of seven homologous protein sequences of other fungi and OhPbs2 was conducted, and the phylogenetic tree was constructed by the maximum parsimony method in MEGA6 to further analyze and identify this gene. Using homologous recombination and protoplast transformation, OhPbs2 was transformed into the ΔCgPbs2 of C. gloeosporioides. The transformant was screened on PDA+1.5 mol·L^-1 sorbitol. At the same time, the genome of the transformant was extracted as a template and identified with the primer pairs of OhPbs2, and the correct transformant ΔCgPbs2+OhPbs2 was selected for subsequent phenotypic determination. The growth state of ΔCgPbs2, ΔCgPbs2+OhPbs2 and wild type strains was compared under different culture conditions. And the pathogenicity of the three strains was detected by inoculating the leaves of the rubber tree.【Result】The full-length of the OhPbs2 is 1 927 bp, cDNA of the OhPbs2 is 1 860 bp, and contains an intron that encodes a 619 amino acids protein. Bioinformatics analysis showed that the protein had the same S_TKc domain as CgPbs2. The phylogenetic tree showed that the Pbs2 protein was closely related to Aspergillus fumigatus Pbs2 protein, and the similarity was 55%. And the similarity to Colletotrichum gloeosporioides was 49%, also it was close to Pbs2 protein of Neurospora crassa, Magnaporthe oryzae, Saccharomyces cerevisiae and Fusarium graminearum, the similarities were 54%, 53%, 53% and 50%, respectively. ΔCgPbs2+OhPbs2 strain could grow white colonies on PDA+1.5 mol·L^-1 sorbitol medium, but ΔCgPbs2 strain could not grow. The sequencing results showed that the OhPbs2 had been successfully transferred into the ΔCgPbs2. The color of ΔCgPbs2+OhPbs2 in the MM medium colony was white with short aerial hyphae, which is different from the wild type strain. The growth rate of ΔCgPbs2, ΔCgPbs2+OhPbs2 and wild type strains decreased gradually with increasing concentration in MM medium containing different concentrations of NaCl, sorbitol, SDS, H₂O₂ and fludioxonil. OhPbs2 not only restored the ability of the wild type strain to tolerate osmolality, especially to sorbitol, but also to the susceptibility to fludioxonil, and even to enhance potency. But OhPbs2 complementary changed the color of C. gloeosporioides and inhibited the growth of aerial hyphae. CgPbs2 might be involved in the pathogenicity of C. gloeosporioides, however OhPbs2 did not restore its pathogenicity, but weakened its pathogenic ability to a certain extent.【Conclusion】OhPbs2 may be involved in the regulation of the vegetative growth, oxidative stress, osmotic pressure and cell wall formation of the pathogen, and enhance the corresponding function of the C. gloeosporioides. OhPbs2 may be involved in the pathogenicity of the pathogen, but pathogenicity may be different from CgPbs2.

Key words: Oidium heveae, OhPbs2, gene cloning, osmotic pressure, pathogenicity