The Role And Molecular Mechanism Of OsmiR1861k In The Resistance To Bacterial Blight Of Rice

The bacterial blight of rice is one of the main rice diseases which can make huge damage to rice production of world. The exploration of related resistance mechanism has become the key step to improve the rice resistance to bacterial blight. MicroRNAs (miRNAs) have emerged as ubiquitous gene regulators in post-transcriptional level, not in transcriptional level, translational level and post-translational level as traditional regulatory factors. Moreover, a large number of studies confirmed that miRNAs play important roles in plant growth, development, and response to biotic and abiotic stresses. The research to action mechanism and biological function of related miRNAs could provide a new view and strategy to improve the rice resistance to disease.On the basis of the gene chip data and protein two-dimensional electrophoresis of a new disease-resistant rice variety SH5, we predicted by bioinformatics analyses that OsmiR1861k was related to rice resistance to bacterial blight. Here, the correlation between OsmiR 1861k and the resistance to bacterial blight was verified by quantative real-time PCR. The OsmiR1861k expression in different rice varieties inoculated with different bacterial blight strains was also analyzed. The predicted target genes of OsmiR1861k were screened through expression patterns, and verified the cleavage sites in vivo. Related functional expression vectors were constructed and transformed into rice. The main results of this study are summarized as follows:(1) The results detected by quantitative real-time PCR showed that precursor and mature OsmiR1861k was up-regulated significantly (p<0.01) in a resistant variety SH5 at 3d and 5d after inoculated by Zhe173, and higher significantly than in a susceptible variety 8411. Thus, we speculated that OsmiR1861k could be associated with the rice resistance to bacterial blight.(2) The expression changes of OsmiR1861k in different rice varieties attacked by different bacterial blight strains were analyzed by quantative real-time PCR. The results showed that, OsmiR 1861k also involed in the resistance to Xoo strains Zhel73 of other resistant varieties besides SH5. The expression of OsmiR1861k in SH5 was significantly (p<0.01) higher than that in 8411 at 5dpi inoculated with P3, P4, P5, P7 and P8 strains. Thus, up-regulation of OsmiR1861k induced by bacterial blight was neither rice variety-specific nor strain-specific. Moreover, the transient overexpression of pre-OsmiR1861k did not lead to hypersensitive response-like cell death in Nicotiana benthamiana, which indicated that OsmiR1861k may involve in the basal defense response.(3) The expression patterns of predicted target genes of OsmiR1861k were analyzed by quantative real-time PCR. Two candidate target genes (Os03g18710 and Os06g44970) were screened out, then their cleavage sites were examined by RLM-5’RACE in vivo.(4) The plasmid constructs for rice transformation including pre-OsmiR1861k overexpression vector, pre-OsmiR1861k promoter-GUS vector and OsmiR1861k STTM silencing vector, were successfully produced. And one pre-OsmiR1861k overexpression transgenic line was obtained. These will contribute to determining the biological funtions of OsmiR1861k by transgenic plants.