Cloning And Functional Analysis Of Strip Rust Resisitance Related Genes From Wheat(Triticum Aetivum.L)

Yellow rust of wheat, caused by Puccinia striiformis f.sp. tritici (Pst),is one of the most destructive fungous diseases in the world. It shows that developing and utilizing the disease resistant cultivars is the most economic, effective and environment friendly method to control the wheat yellow rust. T. aestivum-H. villosa 6VS/6AL translocation lines, developed by the Cytogenetics Institute, Nanjing Agricultural University, shows durable and broad-spectrum resistance to the yellow rust, and the resistance was contributed by the single dominant gene Yr26 located on the chromosome 1B. The Yr26 has been widely used in China, and many wheat cultivars containing this gene have been released and cultivated in the disease seriously attached regions. So, cloning the Yr26 and important defense genes involved in the resistance pathway will facilitate the disease resistance breeding and be valuable to investigate the mechanism of the yellow rust resistance. However, due to special features of hexaploid wheat with large and complex genome and Yr26 located near the centromere region, it was difficult to clone Yr26 using the map-based cloning strategy. In this study, the yellow rust resistance related genes were screened using the Affymetrix Wheat Genome GeneChip, in silico chromosome localization and comparative genomic research, and the function of some interesting genes were comprehensively analyzed by using BSMV-VIGS and gene overexpression. The main results obtained were as follows:1. Preparation by screening the related of theyellow rust resistance gene Yr26The transcript profiles of the resistant 92R137, resistant Y263 and susceptible Yangmai158 was obtained using genechip hybridizatiom. The samples of each material including the Pst inoculated types. Gene expression in leaves of resistant 92R137, resistant Y263 and susceptible Yangmai158 inoculated with CY32 in different times was profiled with Affymetrix Wheat Genome GeneChip. A total of 591 genes were differentially expressed in resistant 92R137 and Y263 with susceptible Yangmai158 after induced by CY3212h. Among them,278 genes were up-regulated and 313 genes were down-regulated. A total of 957 genes were differentially expressed in resistant 92R137 and Y263 with susceptible Yangmai158 after induced by CY32 36h. Among them,299 genes were up-regulated and 658 genes were down-regulated.Using the Blast2go software, it was found that 128 up-regulated genes and 130 down-regulated genes had GO assignments in resistant 92R137 and Y263 with susceptible Yangmail58 after induced by CY3212h; 135 up-regulated genes and 305 down-regulated genes had GO assignments in resistant 92R137 and Y263 with susceptible Yangmai158 after induced by CY3236h. In order to determine the relationship of the differentially expressed genes with Yr26, these differential expression genes were chromosomal localized in silico and comparative genomics analysis with the Yr26 homologous segment of Brachypodium distachyon.A total of 22 up-regulate genes and 29 down-regulate genes were mapped in wheat homoeologous group 1,25 up-regulate genes and 43 down-regulate genes were mapped in Yr26 homologous segment of Brachypodium distachyon,4 up-regulate genes and 7 down-regulate genes were mapped in both wheat homoeologous groupl and Yr26 homologous segment of Brachypodium distachyon in resistant 92R137 and Y263 with susceptible Yangmail58 after induced by CY3212h; A total of 19 up-regulate genes and 68 down-regulate genes were mapped in wheat homoeologous group1,32 up-regulate genes and 104 down-regulate genes were mapped in Yr26 homologous segment of Brachypodium distachyon,2 up-regulate genes and 25 down-regulate genes were mapped in both wheat homoeologous group1 and Yr26 homologous segment of Brachypodium distachyon in resistant 92R137 and Y263 with susceptible Yangmai158 after induced by CY32 36h.2. Cloning and characteristic analysis of two resistane related genes of Yr26Two genes were selected as the target genes in this study according to the Microarray,in silico chromosome localization and comparative genomic research data. These genes showed differentially displayed patterns bewteen the samples of the inoculated resistant material and inoculated susceptible materials. In silico cloning and RACE methods were used to clone the full length genes, and the structure and expression patterns of these genes werealso analyzed in detail.The homologue of Ta.1 4847.1. Al_at, annotated as an F-box protein, was obtained by in silico cloning, and this gene was named as TaFBK, which encodes an F-box protein in N terminal and three Kelch domains in C terminal. The full ORF length of TaFBK is l,062bp and encodes a 353 aa protein. There are 3 members of TaFBK family, and all the members has no introns in wheat genome. The homologues were located on the homoelogous group 1 chromosomes by using comparative genomics analysis with the Yr26 homologous segment of Brachypodium distachyon(TaFBK-A、TaFBK-B、and TaFBK-D).Sequence analysis showed that the TaFBK-B was existed in resistance wheat varieties specially. The qRT-PCR and cDNA sequencing results showed that the expression of TaFBK was induced in three resistance wheat varieties contained Yr26 by Pst, but not expressed in Yangmai158. Additionally, TaFBK was sensitive to MeJA and H2O2, and therefore, it may play a role in the defense resistance to Pst by the signal pathway of MeJA.The homologue of Ta.27275.1.Sl_at, annotated Lr10-like resistance gene, was obtained by RACE, and this gene was named as TaRPMl, which was homologues with BdRPP13.Ta RPP13 is 3,148 bp in length with a 2,826 bp ORF, encodeing a 941 aa protein a 99 bp 5’UTR and 223 bp 3’UTR,which contains a signal peptide, an NB-ARC domain and an SCOP domain.The gene was located on the chromosome 1D using NA/TA of Chinese Spring. The qRT-PCR and cDNA sequencing results showed that the expression of TaRLP1.1 was induced in three resistance wheat varieties contained Yr26 by Pst, but not expressed in Yangmail58.lt is Speculated that this gene was involved in the defense resistance to Pst pathway of Yr26.3. Functional characterization of resistance related genesThe function of the TaFBK and TaRPMl were further characterized comprehensively using the BSMV-VIGS technique and the gene transformation approach. The results showed that the incompatible interaction between the resistant 92R137 and the CYR32 changed to the compatible interaction (infection type4-6) after the TaFBK and TaRPM1 were silenced and further histological observations supported the results. The results indicated that TaFBK and TaRPMl play a more important role in the defense response.Over-expressin of TaFBK-D did not enhance the tolerance to CYR32 in transgenic wheat, however when the TaRPM1 was transformed into the susceptible wheat variety Yangmai158, the characterized transgenic plants showed highly increased resistance to the Pst, and the hypersensitive response was obvious in the interaction sites. At the same time,there are susceptible plants in transgenic wheat of anti-TaFBK-D and anti-TaRPMl.So, it was proposed that the TaFBK-B contributed importantly to the hypersensitive response during the interaction between the host and the pathogen via the MeJA signal pathway and the TaRPMl contributed importantly to the hypersensitive response during the interaction between the host and the pathogen maybe via other signal pathways.