Analysis Of 13 Magnaporthe Grisea Genes Expression Profile And A Preliminary Functional Investigation On OsBTB, A Transcription Factor Gene Induced By M. Grisea In Rice

In the previous study, 12,270 ESTs were generated which were 3 -end sequenced from a cDNA library of rice leaf tissues infected with an incompatible isolate of Magnaporthe grisea, 103(PO6-6) at 2, 4, and 8 h after inoculation and 5,741 unigenes were obtained. The unigene sequences were compared to the draft sequences of the rice and M. grisea respectively. Out of the 5,741 TUTs, 13 TUTs were aligned only with M. grisea genome sequence (http://www.genome.wi.mit.edu) (E-value≤e^-15) using BLASTn algorithm. A cDNA array containing 2,171 unique genes from rice leaf cDNA library was prepared to compare analysis of expression profiles of incompatible/compatible interactions between a pair of blast resistance near-isogenic rice lines H7R/H7S with race ZB15 at 8 h after inoculation, unknown gene set was obtained and were analyzed further by bioinformatics, a gene (clone No. J001E09) encoding a protein containing BTB domain and named OsBTB. Southern blot and Northern blot were performed respectively in order to conform in silico results of 13 putative M. grisea genes and their possible biological function in M. grisea pathogenicity. Full-length cDNA of OsBTB were generated using specific primer and sequencing.Southern blot analysis of 13 TUTs from M. grisea and two ESTs from Oryza sativa in genomic DNAs isolated from M. grisea mycelia and rice leaf, respectively. Genomic DNA (5μg) was digested with restriction enzymes (EcoK I) and subjected to Southern blot analysis. Hybridizations were performed using probes corresponding to the EST sequences of the 15 cDNAs labeled by random priming. Southern blot hybridization assay was carried out by using the 13 M. grisea ESTs and 2 rice ESTs as probes, the results confirmed that these genes originated from M. grisea. Bioinformatics analysis suggested that these genes were imported to pathogenesis-related development. Some of these genes, such as Cyclophilin (GenBank accession No. BI808723) and P-tpye ATPase (BQ907640), were involved in appressorium formation. C-4 methyl sterol oxidase (BI813122) and delta (24)-sterol C-methyltransferase (BQ907428) were involved in the synthesis of ergosterol which is essential for pathogenicity of M. grisea. Plasma membrane sodium response 2 (BI812033) may be involved in signal transduction during the infection. Ribosomal protein L12 (BM420098) and elongation factor 2 (BQ907452) were important to protein synthesis. Coatomer (BI813165) was attached to the trafficking of proteins and secretion within eukaryotic cells. CipB protein (BQ907177) was involved in fatty acid biosynthesis. All the above genes may be involved in host-pathogen interaction in rice tissue. A gene encoding serine/threonine kinase (BQ908235) was involved in conidiaproduction. All the above genes may be involved in host-pathogen interaction in rice tissue. Northern blot assay indicated that these genes were expressed at different degree during appressoria. All genes were not detected during mycelia stage except for cyclophilin. Among these, Plasma membrane sodium response 2 and Nebula related protein were only expressed during young appresorium stage;two genes requiring for ergosterol synthesis were similar to each other;the gene encoding serine/threonine kinase was decreased during appresoria stage. All of these reflected not only their biological functions, but also their abundancy.Specific primers were designed according to in silico full-length sequence of OsBTB obtained, the sequence in the clone J001E09 was amplified by PCR prepared from a rice cDNA library as template. The 1,975 bp long sequence phage DNA contained an open eading (ORF) of 1,677 bp was retrieved through sequencing and assembling, which predicts to encode a protein of 558 amino acids a conserved BTB domain. A DNA sequence about 5174 bp was found through BLASTn to rice genome sequence, containing 6 exons and 5 introns. TATA box and CAAT box were at -27 bp and -HObp on the upstream of transcripton initiator respectively. The similarity was 73.34% between OsBTB and GMPOZ, a nuclear protein, after homology search.This gene was mapped on chromosomes 2 by RFLP analysis using 247 F8 population of Zhenshan97B/Miyang46.The polyclonal antibody was obtained using recombinant OsBTB protein expressed in and purified from E. coli as antigen that was immuned in Xinxilan rabbit. Western blot assay suggested that the expression profiles of OsBTB protein were polyphyletic during different rice lines and corresponding M. grisea isolates incomparible/comparible interaction. There was no specific subcellular location after gold labelled immunosorbent assay. OsBTB and nine known genes (such as Pi-ta, Pib, glucosidase, glycine-rich protein, chitinase, beta-l,3-glucanase, PAL and ubiquitin) were detected using quantative real-time RT-PCR assay, trend of expression in OsBTB was similar to Pi-ta, Pib, chitinase and glycine-rich protein, expression were induced rapidly after M. grisea infiltration, had a peak value at 12h in H7R, but expression of OsBTB reached a peak at 24h. All of genes except alpha-glucosidase were induced after M. grisea infection and reached a peak at 24h in H7S. In addition some genes are induced more rapidly in the resistant than in the susceptible cultivar. RNA interference plant expression vector pCAMBIA-HAN-Cte57B was construced, through partical gun-mediated transformation of japonica rice varieties Zhonghual 1 obtained 1 transgenic plant. PCR screening of the regenerated plant showed that itwas positive. Its biological function was in progress.