Function Of CmWRKY15-1,a Defense Related Gene Of Chrysanthemum White Rust

Chrysanthemum morifolium is a perennial herbaceous flower of compositae and chrysanthemum,which is a traditional flower in China.It has high ornamental and economic value.However,drought,high temperaturefreeze injury,high salt and other adverse stress will affect its growth and development,even lead to death in the complex and changeable growth process of chrysanthemum.Chrysanthemum white rust is one of the most important diseases of chrysanthemum,especially in the cultivation with low temperature and high humidity.The plant disease is more serious,which seriously affects its yield and quality.In recent years,it is common to change the resistance of plants in the molecular level with the development of plant genetic engineering technology.Therefore,it is an effective way to cultivate new varieties with stress resistant by finding the resistance genes of chrysanthemum.Tranfering the specific genes into plants through plant genetic engineering,and carrying out transgenic breeding.In this study,we used the CmWRKY15-1 gene sequence cloned and the recombinant expression vector which gained in the early stage,and constructed a interference vector.Then we transformed them into chrysanthemum to achieve the overexpression and silencing of the CmWRKY15-1 gene.Through a series of functional verification of transgenic plants.we verified the function of CmWRKY15-1gene in resistance response to chrysanthemum white rust,which provided reference for explaining the molecular mechanism of resistance to chrysanthemum white rust,breeding new varieties with resistance gene and improving the ornamental quality of chrysanthemum.The research contents and results are as follows:?1?The CmWRKY15-1 gene expression vector p BI121-CmWRKY15-1 was transformed into chrysanthemum variety ‘Huangying' by Agrobacterium mediated method.The leaves were infected with agrobacterium EHA105 solution containing recombinant plasmid,and we established the efficient regeneration system and screened the antibiotic concentration.The average number of buds of ‘Huangying'was the highest,and the germination rate was 93.3% in MS + 0.5 mg·L^-1 NAA + 0.3mg·L^-16-BA medium.The optimal concentration of cefamycin was 200 mg·L^-1.The optimal concentration of Kan resistance marker screening was 20 mg·L^-1.and the optimal genetic transformation system was: pre-culture for 2 d,bacteria concentration is OD₆₀₀=0.5-0.6,the dilution factor is 50 times and infected for 7min,thenco-cultured for 2 d and delayed time for 2 d,and then 18 Kan resistant plants were obtained.?2?PCR results showed that 8 of the 18 resistant plants were PCR positive,which initially confirmed the transfer of CmWRKY15-1 gene.q RT-PCR showed that CmWRKY15-1 gene could be highly expressed in the identified plants,and the semi quantitative results were consistent with the results of q RT-PCR,which showed that CmWRKY15-1 could be normally retrotranscribed in the transgenic plants and successfully expressed.?3?We successfully constructed the CmWRKY15-1 gene interference expression vector,named RNAi-CmWRKY15-1.And transformed it into chrysanthemum variety‘Huangying' by Agrobacterium mediated method.We used the best selected genetic transformation system and obtained a total of 20 transgenic plants by genetic transformation.8 of them were PCR positive,q RT-PCR showed that the expression of CmWRKY15-1 gene could be effectively inhibited.?4?In phenotype identification and disease index investigation of RNAi-4 lines.The disease index of RNAi-4 lines was 53.67,and its host response was susceptible?S?.However,there was no change in wild-type plants after inoculation,which indicated that the interference of CmWRKY15-1 gene decreased the resistance to white rust.?5?After inoculation,the content of endogenous SA in OE-9 lines increased.By analysising the expression characteristics of SA synthesis gene,we found that the overexpression of CmWRKY15-1 gene promoted the expression of key genes of SA synthesis pathway,and the silencing of CmWRKY15-1 gene repressed endogenous SA and inhibited the expression of key genes of SA synthesis pathway.Which is suggested that CmWRKY15-1 may be involved in the regulation of SA synthesis in response to chrysanthemum white rust.Through the analysis of the expression characteristics of genes responding to SA resistance signals,it was found that the expression of four PRs genes in OE-9 lines was higher than that in WT,especially PR2 and PR5 genes,while the expression of four PRs genes in RNAi-4 lines was lower than that in WT.That is to say,the silencing of CmWRKY15-1 gene can inhibit the expression of NPR1,PR1,PR2 and PR5.At the same time,the silencing of CmWRKY15-1 gene also reduced the expression of PAL,CHI and other disease resistance related defense enzymes.