Construction Of A Mutant Library Of Ustilaginoidea Virens And Cloning And Functional Characterization Of HOG1Gene

Rice false smut, whose causal agent is Ustilaginoidea virens, is an important disease in rice grains. It was reported that the growth, development and pathogenicity were regulated by cellular signal transduction in many fungi, MAP kinase signal transduction pathway, a universal pathway, have an important role in the ability of regulating growth and development processes. To identify the fungal growth, pathogenicity genes and to understand their molecular mechanisms for development and pathogenicity are important for the control of rice false smut. By setting up Agrobacterium tumefaciens-mediated transformation(ATMT) technique, we obtained two growth defect mutant T18and T39, identified a T-DNA tagged gene of T18and primarily demonstrated its function. Furthermore, we Cloned and analysed Hogl MAPK homologous gene UvHogl from U. virens. Main results in this paper were as follows:1. We investigated the expression stability of5Ustilaginoidea virens endogenous candidate genes (18S rRNA, GAPDH, ubiquitin, β-actin and a-tubulin) in real-time quantitative PCR experiments. The analysis with geNorm program revealed that β-actin and a-tubulin could be used as the reference genes for normalization of real-time quantitative PCR experiments data.2. A high-effective Agrobacterium tumefaciens-mediated Ustilaginoidea virens transformation system was established through optimizing various transferring factors. Adopted this transformation system an T-DNA insert mutant library containing about1000transformants was established, this work will provide the foundation for study the function genes of U. virens. We selected two growth defect mutant T18and T39, and found that they have obvious defects in Growth Speed, Sporulation quantity, germination rate compared with wild-type. In addition, we obtained a1800bp flanking sequence by TAIL-PCR, result showed the T-DNA insert in the Promoter of cytochrome c oxidase polypeptide V, but the functions of this gene should be further study.3. Degenerate PCR primers were designed according to the conserved amino acid sequence of several filamentous fungus MAPKs which were homologous to yeast Hog1MAPK, and partial DNA fragment encoding a MAPK was amplified from U. virens using PCR. Then, a whole DNA sequence encoding the MAPK, designated as UvHogl, was obtained from U. virens by extending upstream and downstream sequence of the amplified fragment using RACE method. Sequence analysis suggested that UvHogl encoded a protein of358amino acids with Mr of40.98KDa and PI of5.63. The UvHogl contained the conserved TGY activation loop found in the stress-activated protein kinase subgroup of MAPKs and its amino acid sequence showed95%,93%,88%and81%identity to MgOsm1(AAF09475.1)from Magnaporthe grisea, BbHogl (AAS77871.1) from Beauveria bassiana, AfOsm1(XP752664.1) from Aspergillus fumigatus and CrHogl(AAM26267.1)from Cryptococcus neoformans var. neoformans, respectively. Phylogenetic clustering suggested that UvHogl is homologous to yeast Hogl MAPK.4. The experimental results show that the expression of UvHogl is the same under the low and high NaCl condition and in low NaCl treatment the expression is higher. We also set up a system of preparation the protoplast of Ustilaginoidea virens and construct plasmids for knocking out UvHOG1gene, which would provide a basis for further study on functions of UvHOGl gene.