| Northern corn leaf blight (NCLB), caused by Exserohilum turcicum (Pass.) Leonard et Suggs, is a prevalent foliar disease occurred in most maize (Zea mays L.) production regions worldwide. Yield losses of susceptible cultivars due to occurrence of NCLB can be as high as 30% when the disease is severe. The most effective measure for controlling NCLB is application of host genetic resistance. Since 1961, six do minant genesHt1,Ht3 , HtN,HtM andHtP have been reported. Understanding of resistance to NCLB conferred byHt genes is important for improving maize against the disease.However, the information of the molecular characters ofHt genes orHt-relative genes is not available. This study was carried out to identify theHt2-related genes in RNA level and to provide the basis on studying geneHt2 and understanding the molecular mechanism of resistance to north corn leaf blight. The main results were as follow:1. Using single spore isolating technique, 48 E.turcicum strains were purified from northeast provinces of China and the physiological races were analyzed by using differential host. Fifteen physiological races including 0,1,2,3,N,12,13,1N,23,2N,3N,12N,123N,123,23N were found. Race 0 and 1 were identified more frequently than others and the ratio of N and 1N are increasing, but there are no obvious do minant races in northeast provinces of China. About 41.7 percent E.turcicum can infect corn with one geneHt and 29.1percent, 10.4 percent and 2.1percent can infect corn with two, three and four geneHt respectively.2. Based on the biological assay in vitro, the virulence of 48HT-toxins were identified. The result of virulence analysis fundamentally agreed with that of physiological races identification. At the same time J15, J22 andH7HT-toxins which induced necrotic spot on leaves ofHZS andHZSHt1, but no spot was present onHZSHt2 were selected. J13 and L5HT-toxins induced necrotic spot on leaves ofHZS andHZSHt2, but no spot was present onHZSHt1 were also selected.3. cDNA-AFLP reaction system of corn was established. Seventy out of 1024 prime combinations with clear polymorphic AFLP fingerprints were screened4. cDNA-AFLP was conducted to analyze differential expression ofHt2-related genes between near-isogenic lines (NILs) Huangzaosi (HZS) andHuangzaosiHt2 (HZSHt2) under crudeHT-toxin treatment. Twenty-one transcript-derived fragments (TDFs), designatedH1 toH21, were specifically expressed inHZSHt2. BLAST analysis through the GenBank database indicated that the 21 TDFs identified can be separated into 4 groups. Basal energy metabolism gene and Signal transduction gene expressed at 3 h after inoculation. Transcription factor related gene and part of defiance gene expressed at 3 h after inoculation. Few genes expressed between 12 to 72 h. A gene cluster was found on bin 5.03 by MaizeGDB Blast analysis. The full length cDNA ofH8 (H8 cDNA, GenBank accession number FJ600319) was cloned and sequenced. RT-PCR analysis demonstrated thatH8 was up-regulated inHZSHt2 only at 6 h after inoculation withHT-toxin. The deduced protein encoded byH8 cDNA is 77% similar to Plus-3 domain containing protein which is found in yeast Rtf1.the the mechanism of geneHt2 regulate the resistance toHT-toxin is may similar to translation elongation factor.5.eventy-six TDFs, designated Ex01 to Ex76, were specifically expressed inHZSHt2 by cDNA-AFLP analysis between near-isogenic lines (NILs)Huangzaosi (HZS) andHuangzaosiHt2 (HZSHt2) after inoculation with race 1 of Exserohilum turcicum. BLAST analysis showed that 52 of them were homologous to the genes in the GenBank database. The TDFs with significant protein homology were classified into eight functional categories: basal energy metabolism, transmembrane transport protein, defense/resistance protein, protein metabolism, protein of chromosome and proteins of DNA replication and transcription, signal transduction, growing development mediator and ranscription factor. Most of these genes were associated with basal energy metabolism and defence/resistance gene.Analyzed the space-time trait of 52 TDFs showed that those ranges from chromosome 1 to 9. Signal transduction genes expressed at 6 h after inoculation and defense/resistance genes star to express at 48~72 h.Ht2-related gene expression profiling in response to inoculation of E. turcicum were analyzed by means of cDNA-AFLP. The identification ofHt2-related genes in the present study are important to further study ofHt2 gene and the mechanism of resistance to E. turcicum in maize.6. A full-length QM-like cDNA (designated ZmQM) was cloned from maize (Zea mays L.) leaf tissues using cDNA amplified fragment length polymorphism (cDNA-AFLP) and rapid amplification of cDNA ends (RACE) techniques. The expression of ZmQM was exa mined in leaves of theHt2 isogenic linesHuangzaosi andHuangzaosiHt2 carrying geneHt2 for resistance to northern corn leaf blight after inoculation with race 1 of Exserohilum turcicum. The nucleotide sequence of ZmQM shared 66–92% identity to QM genes isolated from other species. RT-PCR analysis showed that the expression of gene ZmQM was up-regulated inHuangzaosiHt2 at 12 h after inoculation with race 1 of E. turcicum and at 6 h after inoculation withHT-toxin compared withHuangzaosi. the mechanism of geneHt2 regulate the resistance toHT-toxin or E. turcicum by up-regulated QM-like protein (ZmQM) and then active the pathway of QM-like such as NIK-mediated antiviral signaling pathway.7. For further function analysis on the ZmQM, the expression vector containing ZmQM were constructed. |
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