| DON (deoxynivalenol), a virulence factor, plays an important role in the infection of Fusarium graminearum in wheat. The infection ability of F. graminearum depends on its capacity of producing DON. Wheat tissue showed typical FHB symptom when treated with DON only. The production of DON by F. graminearum is significantly decreased in the wheat varieties with scab resistance. At present, several genes related to DON resistance have been isolated from various plants. In order to better understand the molecular mechanisms of FHB or DON resistance, it is critical to identify new genes that are expressed closely related to DON treatment, because the inheritance of FHB resistance and DON resistance is a complicated quantitative trait and the mechanisms under FHB resistance have not yet been clarified.In order to reveal the gene expression patterns of wheat induced by DON and clone genes related to DON resistance, a cDNA library of spikes of scab-resistant wheat Wangshuibai induced by DON was constructed with SMART cDNA library construction kit. The titer of the primary library was 3×106pfu/mL, and the titer of the amplified library reached at 2×109 pfu/mL. The inserted fragments size of the positive clones was 1053 bp at average. The library was stored in 19 mixing pools. In the present reseach, we improved the PCR-based procedure of screening phage cDNA library. The key step in screening library, ’plate-division’ was replaced by’partition in liquid’, which avoids tedious plating, infection and washing procedures. This modification not only decreased the workload of library screening, but also improved the speed and efficiency of obtaining the positive clones.In this study, GeneChip analysis indicated that an EST encoding an ABC (ATP-Binding Cassette) transporter was up-regulated by 45 times in a wheat landrace Wangshuibai, which is resistant to DON accumulation. A pair of EST-derived primers was designed based on the EST sequence, and a clone was then isolated from a wheat genomic DNA TAC library. The TAC clone was sequenced using chromosome walking and gene prediction was conducted using Softberry. A cDNA clone of this gene was subsequently isolated from Wangshuibai induced by DON using gene-specific primers designed according to the untranslated sequence of the gene. The genome size of the gene is 7377 bp, consisting of 19 exons with coding sequences of 4308 bp. It encodes a protein with 1435 amino acid residues and the calculated molecular weight is about 161 kDa. BLAST analysis indicated that the gene may belong to PDR (pleiotropic drug resistance) sub-family, and hence designated as TaPDRl (Triticum asetivum Pleiotropic Drug Resistance). TaPDR1 was located on chromosome 5A of wheat using nullisomic-tetrasomic lines of Chinese Spring. TaPDRl was up-regulated by both DON and F. graminearum. Expression patterns of TaPDR1 were different in wildtype Wangshuibai and the fast-neutron induced Wangshuibai mutant lacking FHB1, a major QTL of FHB resistance and DON resistance in chromosome arm 3BS. These results suggested that TaPDR1 might be a candidate gene responsible for DON accumulation resistance. The expression profile showed that TaPDR1 expression was neither induced by hormones typically involved in biotic stress, such as JA and SA, nor by abiotic stresses, such as heat, cold, wounding and NaCl. However, TaPDRl expression was regulated by Al3+ and [Ca2+], indicating that [Ca2+]i might mediate the signal of TaPDR1 expression. A double stranded RNA interference vector was constructed. Preliminary function of the gene was studied by Agrobacterium mediated transformation method into wheat.A pair of primers, TR2, was designed based on the sequence of TaPDR1 and used for amplification in 23 ecotypes of Brachypodium distachyon from different geographical regions. Detailed sequence analysis showed that the PDR1 genes were highly conserved in B. distachyon. The sequence similarities from different B. distachyon species were more than 95%. Two CAPS markers, which were designed based on SNP sites found in the PDR1 gene of B. distachyon, could differentiate the PDR1 homologous genes from different B. distachyon genome. Based on restriction site analysis, the PDR1s were classified as E type or H type. From 23 B. distachyon ecotypes,39 PDR1s were identified. All ecotypes had either 1 or 2 PDR1 copies. All but one diploid and tetraploid ecotype had only a single H type PDR1. All but one hexaploid ecotype had both an E type and an H type PDR1. Phylogenetic analysis revealed that each type formed a well-supported cluster. The two PDR1 types appeared to evolve differently. These different evolutionary patterns could indicate a difference in age between the two types or might indicate different mutation rates or selective pressures on the two types. The phylogenetic analysis also revealed that the hexaploid ecotypes shared a genomic origin for their E type PDR1,but there were multiple origins for hexaploid H type PDR1.Overall, the results suggest that tetraploid and hexaploid might be misnomers in B. distachyon and suggest a complex polyploidization history during B. distachyon evolution. |
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