The EST Analysis Generated From The Compatible Interaction Between Wheat And Stripe Rust And The Transcription Gene Physical Map Construction

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. Yield losses caused by stripe rust over a large area can be up to 50%, while individual fields can be destroyed when the disease is badly prevalent. Traditionally, research about wheat stripe rust focused on incompatible interactions, explaining the host resistant mechanism and resistance genes exploitation. More recently, increasing attention has been focused on pathogen factors that promote compatible interactions and disease development in plant tissue.So this research gets a lots of EST sequence by sequencing a compatible interaction cDNA library between wheat and stripe rust. We analyses these sequences by bioinformatics methods and well known the gene transcription mode. The RT-PCR and the qRT-PCR were used to analysis the transcription expression of several genes which homologue to plant and fungi in different stage and tissue. At the same time, VIGS technique was used in verify the gene function in the system of interaction of wheat and stripe rust. The last, we successful constructed a gene transcription physical map of stripe rust. The main results of our work are as follow.1. From 6,002 randomly picked clones that were sequenced from 5' end. 5,793 ESTs with high quality were obtained and clustered into 583 contigs and 2,163 singletons to give a set of 2,746 unique sequences (unisequences). EST similarity search using the BLASTx program was carried out by comparing sequences in the non-redundant protein database of GenBank. About 50% of the ESTs showed significant matches to gene coding proteins with known functions; 20% of the ESTs were similar to gene coding proteins with unknown functions; 30% of the ESTs did not show significant similarities to any sequences in public databases. 52.8% ESTs, which was the largest category, showed the highest homology with plants; 16.3% ESTs showed higher homology with fungi and the category of no hits found accounted for 30%.2. To validate the expression patterns of genes generated in cDNA library of wheat-Pst compatible interaction. We tested some gene generating from the compatible interaction using the SqRT-PCR and real-time PCR. Several stripe rust gene have differential expression pattern in the stage of compatible interaction between wheat and stripe rust. Some genes are up-regulated after the rust infection the wheat. We speculated those gene are house keeper genes. For example, actin and ribosome genes. Those genes are expressed by rust with high level. Other genes only expressed at the end infection stage. No signals were detected at the early stage of rust infection. Those genes maybe express by hausteria of rust controlling the rust to acquire the nutrient from the wheat. At the some time, we didn't detected any expression signal of those genes in the healthy wheat. Both WRIS₆71 and WRIS₁264 genes have no more expression change in compatible and incompabtible interaction of wheat and stripe rust. WRIC₁16, WRIS₂33, WRIS₂972, WRIS₈67, WRIS₁790 genes have high level expression in incompabtible interaction of wheat and stripe rust. Three of genes WRIC₄02,WRIS₃105,WRIS₃974 are expressed with high level in compatible than in incompabtible interaction. For test the function of those genes, VIGS was used and four genes, WRIC₄02-γ-23(C2),WRIC₄23-γ-23(B4),WRIS₃105-γ-23(C4)and WRIS₂972-γ-CY23(A8)have the resistance function and control the incompatible.3. A total of 4,219 Pst unisequences from the Ured, GermUred, and Haus libraries were searched for homologous sequences in the Pgt genome of 392 supercontigs. Of the Pst genes, 1,432 had significant homology (e value < 1.00E-5) to Pgt genomic sequences. The three libraries had different percentages of homologous genes with Pgt. The Ured library had the highest percentage, 54.51%, followed by the GermUred library (51.21%), while the Haus library had the lowest percentage (13.64%). In average, 33.94% of the 1,432 Pst genes had significant homology with the Pgt sequences. The 1,432 Pst genes were aligned to 237 linkage groups corresponding to 237 Pgt supercontigs. The 237 linkage groups ranged from 2,878 to 3,081,398 bp with the most of the linkages from 5.0 Kb to 2.0 Mb. Overall, the 1,432 genes matched 787,413 bp and spanned over 86.55 Mb of the Pgt genomic sequences. Because the majority of the 1,432 different genes were aligned to more than one sequence locus, a total of 4,604 gene loci were obtained. The fold of multiple loci per unique gene was unbalanced among the three libraries with 1.30 for the GermUred library, 1.53 for the Ured library and 10.58 for the Haus library. The number of genes varied from 1 to 153, excluding"Supercontig 392"that contained unassembled sequences, with an average of 19 genes per supercontig. Over 70% of the supercontigs have 20 genes or fewer while only 4 supercontigs (Supercontigs 1, 2, 3 and 17) had more than 100 genes. The genes from the three Pst libraries were unevenly aligned to the Pgt genome. Of the 237 supercontigs, the 712 unisequences were aligned to 134 supercontigs; the 441 unisequences were aligned to 121 supercontigs; and the 279 supercontigs were aligned to 213 supercontigs. The gene density (the number of base pairs per gene) ranged from 1,020 to 209,493 bp with an average of 20,500 bp. The majority of the supercontigs had a gene less than 30 Kb, which may be considered relatively gene-rich region. In contrast, a few supercontigs had a gene in over 60 Kb genome region, which may be considered as relatively gene-poor region. These results indicated that genes expressed in different Pst growth stages tended to be clustered on the genome. To validate the linkage relationships of Pst genes, a total of 84 forward and reverse primers were designed for 42 genes to form 21 pairs. The genes in each pair were selected based on their proximity within 50 Kb in the physic map. Clones that were positively amplified with the first pair of primers resulted from the three-dimensional pooling screening were amplified with the second pair of primers. Of the 21 pairs of genes tested, 12 pairs (57%) were successfully identified in same BAC clones. The results clearly showed that these genes in pairs are truly linked in the Pst genome.