Expression Analysis Of TcLr19 Induced By Puccinia Triticina

Wheat leaf rust caused by Puccinia triticina is a major disease in most wheat-growing areas, its epidemics sometimes causing significant yield losses. Resistant cultivars are the most economic, safely and environmental friendly way for minimizing the losses caused by the disease. As one of the few widely effective genes conferring resistance to leaf rust in wheat, Lr19 was transferred into the wheat genome from Thinopyrum sp. To date, it has great potential to be used for wheat production. TcLr19 induced by P. triticina was analyzed by cDNA-AFLP and EST technique. A primary gene expression profile of the disease resistance in wheat was obtained at 0h, 3h, 6h, 12h, 16h, 20h, 24h, 30h, 36h, 48h, 72h and 96h after inoculation in seedling stage.The study was carried out to detect the differential expression of genes in TcLr19 and Thatcher inoculated with P. triticina isolate 05-21-116③(THTS), by complementary DNA amplified fragments length polymorphism (cDNA-AFLP) analysis. A total of 58 of 224 primer pairs displaied polymophism between TcLr19 and Thatcher with 25.9% polymorphic bands. Polymorphic bands between TcLr19 and Thatcher were grouped into seven different types, and five types of them were supposed to correspond to the disease-resistant gene. Twelve of these fragments were sequenced and analyzed. Blastx analysis showed that eight of the twelve were homology with the known function genes.756 clones of the cDNA library from wheat TcLr19 leaves induced by avirulent isolate of P. triticina were sequenced randomly, and 649 qualified ESTs were acquired and clusterted into 472 unisequences. The BLASTX program was used to search for homologous genes of the unisequences in the GenBank non-redundant protein database. 325 unisequences accounted for 68.9% of all the unisequences showed high homology with the function known genes or ESTs, 115 unisequences accounted for 24.4% of all the unisequences showed high homology with the hypothetical protein, and the others have no significant homology to any sequence in the database. All unisequences were classified, 24.3% of these genes shared high homology with genes with unclear function, and 18.4% genes shared high homology with genes involved in energy and metabolism. 20.8% to genes with functions related to disease defense and signal transduction; and those in the remaining groups to genes involved in transcription, transport processes, protein metabolism, et al, respectively.The expression profiles of 6 differential expression fragments from cDNA-AFLP and 2 signal transduction genes and 3 disease/defence genes from ESTs were analyzed by normalization with Actin gene. The results of the RT-PCR showed that the expression patterns of each gene were inconsistent in wheat leaves, consisting mainly of constitutive expression and inducible expression. Constitutive expression genes have the same expression pattern in the various time points of non-inoculated and inoculated TcLr19. However, the inducible expression genes are expressed earlier than that in non-inoculated and increased expression after inoculation.Resistance related genes obtained were analyzed. Calmodulin-binding protein, leucine-rich repeat receptor-like kinase, serine/threonine kinase protein, Protein kinase Pti1, GTP-binding protein and calmodulin TaCaM2-2 were supposed to correspond to signal transduction of plants. Similar to stem rust resistance protein, stress regulated protein, putative leucine-rich repeat receptor-like kinase, oxidoreductase, 2OG-Fe oxygenase family protein, putative glucose-methanol-choline (gmc) oxidoreductase, ABC transporter, zinc finger protein, glutathione-S-transferase, DnaJ protein family-like protein, putative cytochrome P450, NBS-LRR disease resistance protein and cell death-related protein were supposed to correspond to the expression of the disease-resistant gene.According to the target fragment19-9(168bp) amplified by primer P-AT/M-CAC, 3′RACE was carried out with gene specific primers designed based on target fragment and 3′universal primer provided in the kit. An amplified fragment of 2141bp in length which overlapped with the known 19-9 sequence by 156bp was obtained. Finally, a fragment of 2153 sequence was obtained. This sequence showed homology with putative leucine-rich repeat receptor-like kinase, the signal transduction genes in plants are probably responsible for signal transmission through a cell membrane.The gene expression profiles of TcLr19 induced by P. triticina were obtained by cDNA-AFLP and EST. And the kind of expression genes and expression level during incompatible interaction between leaf rust (P. triticina) and TcLr19 were also obtained. This established solid foundation for understanding resistance mechanism of leat rust resistance genes, screening and cloning resistance genes.