| Maize banded leaf and sheath blight (BLSB) is one of the most devastating fungal diseases worldwide which is mainly caused by Rhizoctonia solani Kuhn. It was an important limitation for breeding resistance variety and disease control without major resistance gene identified in germplasm resources. Because the regμlatory of expression was also reported to enhance the plant resistance, it was emphasized to be another strategy to breed the resistance maize by utilizing those abundant of defense-related (DR) genes. One of the ideal strategies was selected with pathogen-inducible promoter which conditional regμlated the expression of DR genes. So identification of new pathogen-inducible promoter and its key czs-element will be benefited for construction of specific expression system for plant resistant engineering.In our previously studies, the gene expression profiling has been done between maize and different virulent strains YWK62or YWK196which identified about300differential expression genes. In this study, two of up-regμlation genes, GRMZM2G127328and GRMZM2G169240were selected to validate the function of promoter, and to identify the cis-elements response to BLSB pathogen.About2Kb DNA fragment upstream the translation initiation site was cloned into the promoter validation vector pCAMBIA1391which contains the reporter gene GUS. Then the total number of14and10transgenic lines was generated with Agrobacterium-mediated transformation in rice variety Zhonghua11. The tissue dissection of expression show that it was similar expressed as those detected in maize tissue with quantitative RT-PCR. In agree with the inducible expression ability in maize, two promoter have been identified to induce the expression of Gus within24h after inoculation of YWK196.Analysis of the two promoter sequences through PLACE showed that there are a lot of specific gene expression sites in addition to some basic regulatory sites, such as elicitor-responsive element. By using transient assay approach for a series of truncation promoter, we identidied that the fragment ranged from-1416bp to-1086bp contains some pathogen inducible cis-elements for PGRMZM2G127328. Three GT-1elements was predicted in this region from PLACE analysis which is a pathogen-and salt-responsive element and have been validated to response to YWK-196in other projects in our lab. It was selected as candidates of putative target cis-elements. The further data showed that the GT-1deletion promoter has been lost the pathogen inducible ability. Therefore, GT-1was identified as the core pathogen-inducible element of promoter PGRMZM2G127328.Similar truncations had been done for promoter of PGRMZM2G169240, and the results was indicated that the fragment ranged from-1278bp to-1183bp could be induced by YWK196. And there was no known pathogen-inducible element had been found in this region after analysis with PLACE software. It is implied that some novel putative pathogen-inducible cis-elements were existed in this region. Split fragments was separately fused into35S mini derived GFP to screen the novel cis-elements. Finally, a30bp fragment ranged from-1263bp to-1234bp was validated to carry the novel pathogen-inducible cis-element.In conclusion, we totally identified two maize promoters which coμld be activated by inoculation YWK196. And we also validated that GT-1was the core pathogen-inducible element of promoter PGRMZM2G127328, and the pivotal pathogen-inducible region of promoter PGRMZM2G169240should be located on a30bp fragment. These results were provided the research basis to explore the susceptible mechanism and to facilitate the genetic engineering of BLSB-resistant. |
PDF Full Text Request