Genome-wide Analyses Of Cytokinin Responsive Genes And Studies On Molecular Mechanism Of Response Regulators In Rice(Oryza Sativa L.)

Cytokinins are a class of plant hormones that regulate many aspects of growth and development, including cell division, apical dominance, chloroplast biosynthesis, root and leaf differentiation, leaf senescence, nutrient signaling and shoot differentiation et al. In prokaryote and eukaryote, the organisms can sense and respond the external environment through a two-component signaling system (TCS). However, much of knowledge for TCS so far was based on research in Arabidopsis, a dicotyledonous model plant, but few in monocotyledon. To be better understand the role of two component signaling system in rice, we analyzed the cytokinin responsive genes by bioinformatics methods. The expression patterns of TCS components in the transgenic plants with both overexpressed and RNAi for OsRR3-OsRR6 genes were investigated. The results were showed as following:1. By analyses of conserved domains of TCS genes in rice, maize and Arabidopsis genome, we found that most of this genes had highly conserved gene sequence with transmitter domain and received domain, and there was no significant difference among the plant species. But the phylogenetic analysis results indicated that TCS gene evolution in rice and maize were closer than in Arabidopsis. The chromosome distribution of TCS gene in these three plants were similar and there was no hot distribution chromosome. Motif analyses showed TCS genes'motif among Arabidopsis, maize and rice have highly homologous, there is a degree of redundancy may reinforce by the gene duplication.2. The OsRR3-OsRR6 genes were cloned by PCR method, the overexpression vectors of OsRR3-OsRR6 genes were then transformed into rice (Japonica cultivar-group: Zhonghua11) mediated by Agrobacterium. We screened two transgenic rice with overexpressed OsRR3 and OsRR5 genes as research objects by cytokinin sensitivity test. The real time PCR quick examination system of copy number in transgenic plants was established. Finally T3 generation transgenic rice of OsRR3 and OsRR5 overexpression with single copy external gene were obtained.3. The sharp of T3 generation of transgenic rice with overexpressed OsRR3 and OsRR5 genes had no significant difference compared with Zhonghua11 (WT rice). However, after exogenous hormone treatment, transgenic plants showed insensitivity to exogenous cytokinin, both OsRR3 and OsRR5 overexpressors had longer roots, more lateral roots, slow callus formation and chlorophyll content decreased faster after dark induced compared with Zhonghua11.By exogenous cytokinin 6-BA treatment, the expression of most type-A OsRR genes was repressed by OsRR3 and OsRR5 genes overexpression except OsRR2, OsRR4 and OsRR6 genes showed increased expression while OsRR8, OsRR12, OsRR13, OsRR19and OsRR22 genes were not detected. OsRR11 gene was not expressed in leaves but roots. Both OsRR3 and OsRR5 mainly acted as negative regulators of cytokinin signaling, as indicated by the reduced sensitivity of OsRR3 and OsRR5 overexpressors to exogenous cytokinins while type-B response regulators were not induced by cytokinins. The results showed that, in rice two component signaling system, both OsRR3 and OsRR5 played a major role in negative regulation to the other A-type response regulators.4. The type A response regulators OsRR3-OsRR6 interference fragments in rice were cloned by PCR method, OsRR3-OsRR6 RNAi vector were then transformed into rice mediated by Agrobacterium. Finally, transgenic plants with lower OsRR6 expression were selected based on cytokinin sensitivity assay, T3 generation transgenic plants with single copy external gene were obtained identified by real time PCR.5. T₃ generation transgenic rice with OsRR6 gene RNA interference have normal phenotype. The transgenic plants showed more sensitive to exogenous cytokinin than Zhonghua11. It also had longer main root, more lateral roots, faster callus formation, chlorophyll content decreased slowly after dark induced compared with Zhonghua 11.After exogenous cytokinin 6-BA treated for 2 hours, OsRR19, OsRR22 and OsHK4 genes in the roots and leaves were not expressed, OsRR11 only expressed in roots and OsHP4 only expressed in the leaves. The expression of most type-A OsRR genes except OsRR8, OsRR12 and OsRR13 genes were increased significantly in OsRR6i transgenic plants compared with Zhonghua11. In both transgenic and untransformed plants, transcripts of OsHKs were up-regulated by 6-BA in both roots and leaves while OsHPs and type B response regulators were not. It indicated that the interference OsRR6 gene could only regulate type-A response regulators transcription and expression, but had no effects on OsHKs, OsHPs and type-B response regulators. The results verified the OsRR6 played a negative regulator role in rice two component signaling system.