| Maize banded leaf and sheath blight(BLSB) caused by Rhizoctonia solani Kühn is becoming a serious and major disease in main production areas of the world. With the increasing of planting density and promotion of new cultivation technology, it has seriously affected and restricted in the yield and quality improvement of maize in China. However, due to the lack of resistant resources, it is limited to breed resistance varieties by using major R genes. Promoter is a “switch” to influence the expression of genes. So it is an alternative strategy to drive the expression of defense-related genes under the pathogen-inducible promoter.Two pathogen-inducible promoters for R.solani, PGRMZM2G315431 and PGRMZM2G174449, had been analyzed in our previous studies. Three regions with unknown cis elements were identified roughly by a series of 5’-deletions. In this study, two elements were found by similarity analysis of three candidate sequences. GT/CTGA was found in PGRMZM2G174449-490--478. TATT/AT was found in PGRMZM2G174449-574--550. And they were co-existed in PGRMZM2G315431-1243--1228. These two elements are selected as candidates of R.solani-inducible cis-element which are analyzed to involve in the response to R.solani in this study.By transient expression in tobacco using a GFP reporter gene, we found that the element of GTTGA could be responsed to pathogens. It could induced the expression of reporter gene downstream. The further data showed that the deletion fragment in PGRMZM2G315431-1243--1228 has been weaken the pathogen inducible ability, but not completely lost. This indicated that GTTGA was involved in the response to R.solani. Moreover, there was the other cis element which was related in the response pathway existed in PGRMZM2G315431-1243--1228. The conclusion is coincided with previous hypothesis. Besides, the important role of GTTGA in the response to R.solani was also proved in stable expression in transgenic rice. The fluorescence signal was also detected with the point mutation of GCTGA. It was indicated that second site of GTTGA was not the key site of the cis element. In addition, the pathogen inducible abilities in different times of GTTGA were evaluated. Compared with singleton of GTTGA, both tandem repeat and triple-repeat have shown stronger fluorescence signals, while there has no difference between tandem repeat and triple-repeat for their pathogen-inducible abilities. This suggested that the inducible abilities can be strengthened by tandem repeat, but it does not accumulate unlimitedly. Similarly, by transient expression in tobacco, the cis element of TATAT was found to be activated to pathogen. Additionally, the deletion region in PGRMZM2G174449-574--550 lost the inducible ability. These results indicated that TATAT was also a cis element which was able to response to R.solani. Furthermore, the ability was also proved in stable expression in transgenic rice. The fluorescence signal was also detected after the point mutation of TATAT into TATTT. It indicated that this mutation site was not the key site of the cis element.In conclusion, this study identified two new key cis elements, GT/CTGA and TATT/AT, These two cis elements can response to R.solani respectively. These results provided not only the basis to reveal the transcriptional mechanism of defense-related genes regulated by R.solani, but also new resources of synthetic promoters to improve the crops resistance by genetic engineering. |
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