Functional Identification Of Chrysanthemum White Rust Defense Gene CmWRKY15-1 Based On TRV-VIGS

Chrysanthemum(Chrysanthemum morifolium)is a traditional famous flower in China.It has high commodity value and is one of the important agricultural products for exporting foreign exchange.In recent years,the chrysanthemum industry chain has continued to elongate,and it has been deeply integrated with rural tourism and rural revitalization.The cultivation and economic development of chrysanthemum germplasm resources have achieved remarkable results,becoming a new growth point for agricultural economic development.In the process of chrysanthemum cultivation,the facility cultivation environment of low temperature and high humidity easily cause a large-scale outbreak of chrysanthemum white rust,and even cause the death of chrysanthemums,seriously affecting its ornamental quality and causing economic losses.Practice has proved that only by clarifying its disease resistance mechanism and selecting disease-resistant varieties can the occurrence of chrysanthemum white rust be fundamentally prevented.In this study,the resistant chrysanthemum ‘C029’ was used as material,and the chrysanthemum white rust resistant differential expression gene CmWRKY15-1 obtained from the previous cloning was used to construct a VIGS silencing vector and transiently transform the chrysanthemum.To understand its molecular mechanism of resistance and provide theoretical and practical basis for chrysanthemum resistance breeding,we used VIGS silencing vector instantaneously transform the chrysanthemum to achieve effective silencing of the target gene and verified its resistance to white rust in the chrysanthemum.The research contents and results are as follows:1.Tobacco rattle virus(TRV)was used as the vector,and the leaf c DNA of the disease-resistant chrysanthemum ‘C029’ is used as the template,and primers are designed outside the conserved region of the CmWRKY15-1 gene sequence to amplify a 216 bp silent fragment.The fragment has inserted into the p TRV2 vector Eco RI and Bam HI double restriction site to complete the construction of p TRV2-CmWRKY15-1 recombinant vector,and use the freeze-thaw method to transfer into Agrobacterium competent EHA105.2.Using the silencing expression vector p TRV2-CmWRKY15-1,and chrysanthemum tissue culture seedling as the receptor,the chrysanthemum transient expression system was established by the Agrobacterium infiltration method,and a total of 10 effective silent plants were obtained through virus detection,semi-quantitative and fluorescent quantitative detection.The expression efficiency is about 25.1%-51% of the normal level,which providestest materials for the next step to verify the function of the CmWRKY15-1 gene in the resistance to white rust of chrysanthemum.3.In order to verify the function of CmWRKY15-1 gene in the resistance of chrysanthemum against white rust,WT and silent plants were inoculated.After inoculation,the relative expression of SA synthesis genes PAL and ICS1 in WT increased significantly,while the silencing plants of CmWRKY15-1 gene inhibited SA synthesis gene expression,indicating that the stress of chrysanthemum white rust pathogenic bacteria promotes SA synthesis and accumulation.CmWRKY15-1 gene may respond to the white rust of chrysanthemum by participating in the regulation of SA synthesis.Through q RT-PCR expression analysis of SA pathway marker genes,it was found that the expression of NPR1 and PR1 in silenced plants was significantly inhibited,indicating that silencing CmWRKY15-1 would inhibit the expression of SA pathway defense-related genes.