Elucidation Of The MiR164c-guided Gene/protein Interaction Network Controlling Seed Anti-aging Ability In Rice

miRNAs participate in all aspects of plant physiological metabolism.Based on previous studies,miRNAs play an important role in regulating rice seed vigor,and the expression of miR164 c is negatively correlated with seed vigor,but its regulatory mechanism is not yet clear.In this study,the network mechanism of miR164 c in the regulation of anti-aging ability in rice seeds at the transcriptome and proteome levels were explored.The results are as follows:1.There are significant differences in the anti-aging ability of rice Kasalath(wild type)and its MIM164 c and OE164 c mutant seeds.MIM164 c seeds are the strongest,Kasalath seeds are the second,and OE164 c seeds are the weakest.Correspondingly,the transcriptome and proteome of these seeds are also significantly different.2.The results of enrichment analysis of GO,KEGG,Map Man,etc.of differentially expressed proteins and differentially expressed genes,as well as the association analysis of transcriptome and proteome,indicate that they may affect seed vigor through six types of metabolic functions,namely stress response,endoplasmic reticulum protein processing,embryo development,serine endopeptidase inhibitor,energy metabolism and others.Among them,the expression levels of energy metabolism-related proteins and serine endopeptidase inhibitors in MIM164 c and Kasalath seeds were significantly higher than those of OE164 c seeds,while most of the related proteins of stress response,endoplasmic reticulum protein processing,embryo development and other functional types were reported to be associated with stress reaction,their expression levels in OE164 c and Kasalath seeds were significantly higher than MIM164 c seeds.3.With the MERLIN iterative algorithm and String database to perform gene-protein interaction network,it was uncovered that the target genes of miR164 c,PSK5,and TIL1 interact with a ubiquitin-related hub gene RPS27 AA,which then interacted with the proteins among the abovementioned six types of functional proteins.RPS27 AA may be the key node guided by miR164 c in regulating rice seed vigor.4.The result of the transcriptome and proteome was verified through Real-time RT PCR and multi-reaction monitoring mass spectrometry analysis.The expression of four closely interacted core genes RPS27 AA,PSK5,Os J?23767,and Os10G0488100 together with six functional genes related to four types of functions among the abovementioned six were determined in rice Kasalath and its MIM164 c and OE164 c mutant seeds before and after artificially aging.The result showed that the expression levels of RPS27 AA and other core genes were closely related to the anti-aging ability of the three materials.The expression levels of the 6 genes were also significantly affected by the effects of aging.In summary of 1 to 4,miR164 c may affected the expression of the core gene RPS27 AA by regulating the expression of the target gene(also core gene)PSK5,and further affect the expression of six types of functional genes to regulate the rice seed vigor.5.Through the combination of our data with the gene expression data of rice under multiple stresses obtained from the NCBI database,the expression of differentially expressed genes between rice Kasalath and its MIM164 c and OE164 c mutant seeds in different MERLIN co-expression/regulation modules were analyzed,The result showed that many of the key modules mediated by miR164 c were related to the seed anti-aging ability,these modules account for more than 80% of all modules,and RPS27 AA happens to be the regulation factor of module 20 and module22,which are two modules most likely regulating the anti-aging ability of rice seed,the expression of 6 genes of these two modules in response to artificially aging treatment was well verified through RT-q PCR test,which further proves the key role of RPS27 AA in regulating seed vigor.At the same time,it is also found that these modules related to the anti-aging ability of rice seeds were also related to the resistance of rice to pathogen.Above all,our study revealed the complicated gene/protein regulation network of seed vigor guided by miR164 c and centered on RPS27 AA,it is also discovered that miR164 c functions in other kinds of stress resistance response other than ant i-aging including the resistance to pathogen stress,drought stress,salt stress and so on,these results lay the foundation for the follow-up study of the molecular regulation mechanism of miR164 c.