Cloning And Biological Function Analysis Of TuRFE In Triticum Urartu

Wheat stripe rust caused by Puccinia striiformis f.sp.tritici is an airborne fungal disease which occurred in different degrees in wheat growing regions all over the world.Wheat stripe rust is very serious in China,and has become a limiting factor affecting the sustainable development of wheat production.Research and practice at home and abroad have proved that breeding and planting resistant varieties is the most economical,effective and safe measure to control wheat stripe rust.Therefore,researches on the wheat stripe rust resistance mechanism and the interaction mechanism between wheat and stripe rust have always been the research hotspots in plant disease resistance and its area.Because the genome of the common hexaploid wheat is complex and difficult to be transformed by heredity,we choose the Triticum urartu which offer A group of wheat as the model plant.Compared with the hexaploid wheat,the genome is much simplified,whic h is more beneficial to the research and verification of the gene function.Candidate genes were screened by transcriptome analysis,and analyzed by BSMV-VIGS.The main results are as follows:1.By inoculating 147 wild-type materials,they were divided into five classes based on their resistance phenotypes,which were 0(immune),1(have HR response),2(partial necrosis,sporulation),and3(Medium sense),4(high sense),and statistics.One of the CYR33 compatible reaction types of Tu26 and an incompatible reaction of Tu 138 were selected respectively,and the RNA of 0,4 hpi and 24 hpi leaves were extracted and performed transcriptome sequencing to collect,then analyze the differentially expressed genes and enrichment to get the candidate gene Tu RFE.2.The full-length primers were designed to amplify and sequence the Tu RFE gene.BLAST analysis was performed by NCBI.It was found that Tu RFE had a RING-type domain and the gene annotation was E3 ubiquitin ligase,which was subcellularly localized in the nucleus.Quantitative primers were designed and expressed in different tissues of Triticum urartu.It was found t hat t he expression level was highest in roots.The expression of compatible(Tu 26)and incompatible(Tu 138)Triticum urartu were detected after inoculation of CYR33.The time points were 0,4 h,8 h,24 h,72 h,120 h,and the expression of Tu RFE was induced by stripe rust.We also observed the infection structure of Puccinia stripe under two kinds of background.We could see that the growth rate of stripe rust fungi under affinity background is obviously larger than that of non compatible background.3.Using the BSMV-VIGS system to silence the Tu RFE in the different incompatible Triticum urartu,the Tu RFE was silenced and inoculated in three different incompatible background materials.After 14 days,the phenotype was observed,and the resistance of these incompatible materials could be obviously reduced after the comparison with the wild type and BSMV-γ.The leaves of 12 h and 24 h after inoculation were used to detect the expression of Tu RFE gene.The expression of some pathogen-associated genes(PR)was detected at the same time.The expression of these genes was also significantly inhibited.Histological observation of the invasion structure,the hypha growth after silencing was significantly greater than BSMV-γ.The results of reactive oxygen also showed that the active oxygen around the stomata invaded by the stripe rust was significantly reduced.These results all indicate that reducing the Tu RFE gene will cause a decrease in resistance of Triticum urartu to stripe rust.