Identification And Functional Characterization Of Defense-responsive Gene OsDR2 In Rice

As we all know, rice is one of the most important crops in the world. However, the outbreaks of diseases caused by bacteria, viruses, fungi and invertebrates always lower both rice's yield and quality. As a result, in order to control such epidemics and reduce the negative impact caused by pathogenic disease, detailed studies of plant-pathogen interactions and the signaling mechanisms become especially necessary.According to the studying results of Zhou (2002) and Wen (2003), the expression of EI5P11, coming from the normalized whole-life-cycle MingHui63 cDNA library, constructed by Chu (2003), was increased in Oryza sativa L. C101LAC, IRBB4, MH63, containing resistance gene Pi-1, Xa4 and Xa26, respectively, after inoculated by its corresponding incompatible pathogenic isolates. Especially at the third day after inoculation, its expression reaches the top amount. As a conclusion conferred from their studies, the gene containing EI5P11 cDNA perhaps is an element that joins the plants defense reaction. And BLAST result shows that there are many EI5P11 cDNA homologous sequences existing in rice chromosome 1 and 7 (Wen et al., 2003).The purposes of this study are to clone the gene represented by EI5P11 cDNA clone, transform this gene into susceptible and resistance rices, and comfirm its function in defense-responses.According to the latest information publisized in NCBI, EI5P11 is existed in rice chromosome 7, the 3'-end of this cDNA clone is nearly identical (94% identity and [E]=0.0 ) to a part of sequences on the BAC clone AP003846, encoding a putative Nt-gh3 deduced protein, belonging to the firefly luciferase superfamily. And it has many alleles located on difference chromosomes in rice. Because of the relationship between this gene and the defense reaction, we named this gene as OsDR2.Through 3'-RACE (3'-rapid amplification of cDNA end) and RT-PCR (reverse transcription-ploymerase chain reaction) , we confirmed the positions of OsDR2's transcriptional terminator and intron. Moreover, the length of OsDR2 gene's transcriptional region was comfirmed by Northern blotting technology.In this study, a highly susceptible japonica cultivar, Mudanjiang 8 was transformed with the overexpression promoter, ubiqintin, fused to the OsDR2 gene mediated by Agrobacterium to confirm the OsDR2 gene's function in rice's defense responses. At the same time, we used RNAi technology to suppress the expression of this gene in Minghui 63, a resistance indica cultivar.In the overexpression test, part of T0 Mudanjiang 8 plants appeared some abnormaltraits, such as stunted form, growth and development with a slow speed and unbearing. And with the analysis of RT-PCR and Northern blotting, we found that the expression of OsDR2 gene in these abnormal plants were much higher than the normal plants and untransformation ones under the condition of non-pathogen inoculation. From this result, we can confer that this gene has the ability to impact the process of plant's growth and development, at least in rice. However, the expression level in transgene plants showed no relationship with the copy number of transgene.After inoculation ToMudanjiang 8 plants with Xanthomonas oryzae pv. oryzae (Xoo) PXO61, we found that the resistance character had a distinct improvement in the transformation plants. This result confirmed further that OsDR2 gene could participate the defense responses in rice.And the Agrobacterium transformation of OsDR2 gene RNAi suppression expression was underway.