| Wheat stripe rust is one of the most important diseases that threaten wheat production worldwide. Identification of the key genes regulating growth and development of the wheat stripe rust pathogen(Puccinia striiformis f. sp. tritici),and further understanding of the mechanism of wheat-rust interaction,have great importance for designing novel biotechnologies and breeding disease resistant varieties. In recent years,the whole genome sequencing of rust pathogens have been completed,providing an important basis in functional genomics studies. However, due to the strict biographic fungi of wheat stripe rust, and the lack of effective genetic transformation method, progresses on the functional genomics of wheat stripe rust pathogen are limited.Host-induced gene silencing(HIGS) is a newly developed method for the studies on gene functions in pathogens. The expressing in plants of double-stranded RNA of pathogen genes, might induce specific silencing of the target genes in pathogen. With HIGS system, not only can we identify gene functions of plant parasitic pathogens through reverse genetics method, but also can produce transgenic plants with enhanced disease resistance. Several HIGS studies on wheat fusarium head blight and powdery mildew have shown the effectiveness.The cell wall is the essential structure of wheat stripe rust and other filamentous fungi, it can ensure the normal development of the fungi and also participates in plant-pathogen interaction. Therefore, the cell wall synthesis is an ideal target for protecting against fungal diseases. Meanwhile, recent years the complete genome sequencing of the wheat stripe rust has provided good basis for functional genomics in rust pathogen. In this study, the cell wall synthesis related genes-- chitin deacetyl transferase(CDA) of the wheat stripe rust pathogen were annotated and cloned. The expression pattern of CDAs during rust infection was analyzed using RT-PCR. Transgenic wheat plants expressing RNAi constructs of the CDA gene were obtained, laid good foundation for its functional studies n the wheat- rust interaction. The main results are as follows:(1) The annotation of CDA genes in wheat stripe rust pathogen : CDA genes were searched and annotated based on the PST78 sequencing project database. The gene structure and sequence gap were corrected based on PCR and sequencing with cDNA of race CRY32.Finally, a total of 20 CDA genes were confirmed in wheat stripe rust pathogen. Phylogeny analysis can divide the CDA genes into 3 main clades. Two of them contain few tandem duplications of CDA genes.(2) The expression analysis of CDA gene in wheat stripe rust:expression pattern was detected on 9 infection stages on wheat cultivar Huixianhong using RT-PCR. The results showed that the expression levels of CDA gene were decreased significantly after infection 72 hours, and the expression was lowest or not detectable after one week. In addition, there are differences in the expression level or the expression pattern of few different CDA genes.(3) The vector construction of RNAi silencing and wheat genetic transformation:we have cloned RNAi fragments from 19 CDA genes and constructed 23 silencing vectors.Using wheat biolistic method, a total of 13 transgenic lines of CDA genes were obtained.(4) Cloning and verification of pathogen inducible promoter:The sequence alignment and splicing of homologous gene were carried out in wheat based on the barley HvGER4 c gene, and the promoter of TaGER4 c gene was cloned with NCBI # KR817251-KR817253. The GFP- fusion expression vectors were constructed and the genetic transformation of Brachypodium were carried out. Through rust pathogen inoculation,transcription analysis and fluorescence detection of the reporter gene GFP, the results showed that the TaGER4 c promoter, mainly functions in the leaf, and can be enhanced after rust inoculation, thus representing a pathogen inducible promoter. |
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