Preliminary Studies Of Osa-miR439a And Osa-miR164a On The Regulation Rice Immunity And The Agronomic Traits Of Rice

The rice blast disease,caused by the fungus Magnaporthe oryzae,is the most important disease of rice(Oryza sativa).MicroRNAs play crucial roles in plant responses to pathogen infections.In recent years,dramatic studies demonstrate that microRNAs also play a crucial roles in plant growth and development.In order to understand the regulatory mechanism of microRNAs with rice blast fungus,we chosed and studied miR439 a and miR164 a.Initially,we constructed over-expression clone and targets mimicry clone about miR439 a and miR164 a.Moreover,we analyzed the disease resistance and agronomic traits in over-expression and targets mimicry materials.Finally,we detected the expression patterns of miRNAs and target genes in LTH and IRBLkm-Ts materials by qRT-PCR,we also detected the transcription level of target genes in over-expression and targets mimicry materials by qRT-PCR.Taken together,the results indicate that miR439 a and miR164 a not only negatively regulated the resistance of M.oryzae in rice,but also regulated the agronomic traits of rice.Our results and conclusions are as below:(1)Results about the miR439a(1)Analysis of the expression pattern of miR439 a in the LTH and IRBLkm-Ts materials.We found that the miRNA level of miR439 a was significantly up-regulated in the LTH materials after spraying blast fungus,while it was’ t change in the IRBLkm-Ts material after spraying blast fungus.The result indicates that miR439 a was involved in rice immunity against M.oryzae.(2)Identification the blast disease-resistance in overexpressing miR439 a materials and it’s target mimicry materials.we inoculated these materials with rice blast fungus,we found that overexpressing miR439 a materials display enhanced susceptibility to M.oryzae,while the target mimicry materials of miR439 a display enhanced resistant to M.oryzae.These results indicate that miR439 a negatively regulates rice immunity against the blast fungus,while the target mimicry of miR439 a positively regulate rice immunity against the blast fungus.(3)Investigation agronomic traits in the overexpressing miR439 a materials.Compared with control materials,we found that the agronomic traits of overexpressing miR439 a materials became bad.These datas indicate that miR439 a negatively regulates agronomic traits in rice.(4)Detection mRNA levels of miR439 a target genes.Through defecting the mRNA levels of the miR439 a target genes,we found that the mRNA levels of LOC＿Os01g23940,LOC＿Os01g36270 and LOC＿Os04g47820 were down-regulated in overexpressing miR439 a materials,while it were up-regulated in target mimicry materials.The indicates that miR439 a maybe cleave its target genes on the post-transcriptional level.(5)Analysis of the expression pattern of target genes of miR439 a in the LTH and IRBLkm-Ts materials.We found that the miRNA level of predicted target genes of miR439 a were induced with varying degrees in both LTH and IRBLkm-Ts materials after spraying blast fungus.These results indicate that target genes of miR439 a are involved in rice immunity against M.oryzae.(6)Identification the blast disease-resistance in over-expression mutant of LOC＿Os04g47820 materials.Firstly,we detected the mRNA level of LOC＿Os04g47820gene in LOC＿Os04g47820 in the mutants lines.Secondly,we inoculated with M.oryzae.and found that the mutants lines display enhanced resistant to M.oryzae.The result indicates that the over-expression mutant of LOC＿Os04g47820 positively regulate rice immunity against the blast fungus.We speculated that miR439 a may negatively regulate rice blast resistance through target gene LOC＿Os04g47820.(2)Results about the miR164a(1)Analysis of the expression pattern of miR164 a in the LTH and IRBLkm-Ts materials.We found that The mRNA level of miR164 a was down-regulated in the LTH materials and IRBLkm-Ts materials after spraying blast fungus,the result indicates that miR164 a was involved in rice immunity against M.oryzae.(2)Identification the blast disease-resistance in overexpressing miR164 a materials and it’s target mimicry materials.we inoculated these materials with rice blast fungus,we found that overexpressing miR164 a materials display enhanced susceptibility to M.oryzae,while the target mimicry materials of miR164 a display enhanced resistant to M.oryzae.These results indicate that miR164 a negatively regulates rice immunity against the blast fungus,while the target mimicry of miR164 a positively regulate rice immunity against the blast fungus.(3)Investigation agronomic traits in the overexpressing miRmiR164 a materials.Compared with control materials,we found that the agronomic traits of overexpressing miR164 a materials became bad.These datas indicate that miR164 a negatively regulates agronomic traits in rice.(4)Detection mRNA levels of miR164 a target genes.Through defecting the mRNA levels of the miR164 a target genes,we found that the mRNA levels of LOC＿Os04g36880,LOC＿Os04g38720,LOC＿Os08g10080 and LOC＿Os12g41680 were down-regulated in overexpressing miR439 a materials,while it were up-regulated in target mimicry materials.The indicates that miR164 a maybe cleave LOC＿Os04g36880,LOC＿Os08g10080 and LOC＿Os12g41680 target genes on the post-transcriptional level.(5)Analysis of the expression pattern of target genes of miR164 a in the LTH and IRBLkm-Ts materials.We found that the miRNA level of target genes of miR164 a were induced with varying degrees in both LTH and IRBLkm-Ts materials after spraying blast fungus.These results indicate that target genes of miR164 a are involved in rice immunity against M.oryzae.