Functional Study Of Mir529a In Rice (Oryza Sativa L.)

Rice is the main food crop in China,its high production and stable yield is directly related to the national food security.Plant architecture is an important determinant of rice yield,and improving plant architecture,especially breeding ideal plant type has become one of the objectives of modern high-yield breeding.SQUAMOSA Promoter binding protein-Like(SPL)is a plant-specific transcription factor family,which plays a key role in rice plant architecture and is regulated by specific miRNAs.On the other hand,rice often suffers various environmental stresses,such as high temperature,cold,flood,drought,hypoxia,cadmium and high salt,which are detrimental to plant growth and development,and affect rice yield and quality.MiRNA is an important regulatory factor,not only regulates plant growth and development,but also responses to abiotic stresses as a key regulator.MiR529 is a class of non-coding small RNAs,which is highly homologous to miR156,and widely distributed in terrestrial plants.There are two copies of miR529a and miR529b in rice.At present,most of the studies have been focused on miRl 56 because of its homology to miR529 and some common targets.While,the function work on miR529a is relatively little.In this study,we constructed the MIR529a overexpression vector to do rice transformation and get miR529a overexpressing transgenic plants,which were used to elucidate the function of miR529a in plant architecture and environmental stress.The major results are summarized as follows:1.There exists tissue-specific expression of miR529a in rice,which was highly expressed in panicles.While miR529b is not tissue-specific expressed.Five target genes of miR529a were predicted using psRNATarget,namely OsSPL2,OsSPL14,OsSPL16,OsSPL17 and OsSPL18.All target genes were highly expressed in early panicles,but lowly in roots,shoots and leaves.These results implied that miR529a and its SPL targets were preferentially expressed in early young panicles,which might be involved in regulating the development of early young panicles.2.The plants of miR529a overexpression showed semi-dwarfing,multi-tillers;smaller stem diameter,thinner stem wall and stem inner cavity.The miR529a overexpressing plants also exhibited shorter and narrower leaves and smaller leaf angle;thinner and shorter roots,and more lateral roots number;and the number of panicle branches,total spikelet and panicle length were significantly decreased.These suggested that miR529a may play a key role in plant architecture.3.At vegetati-ve stage,only the expression of OsSPL2 and OsSPL14 was significantly decreased in miR529aOE.Simultaneously,the expression of tiller-related genes OsTB1,ARF6 and OsPIN1 were also significantly reduced,showed that miR529a may regulate OsSPL2 and OsSPL14,consecutively affecting the expression of OsTB1,ARF6 and OsPIN1,to control rice tiller.4.At the development stage of young panicles,only the expression of OsSPL2,OsSPL14 and OsSPL17 was significantly reduced in miR529aOE,whereas the expression of OsSPL16 and OsSPL18 was not significantly affected.These indicated that miR529a could regulate the growth of panicle by degradation of transcripts of OsSPL2,OsSPL14 and OsSPL17.In addition,the expression of panicle development related genes OsTB1,OsLAX1,OsIAA1,OsIAA6,OsIAA24,OsSIZl,OsDEP1 and OsCSLD4 was significantly changed in miR529aOE.Therefore,these genes may serve as the downstream genes of OsSPL2,OsSPL14 and OsSPL17 to control the development of panicles.A total of 117,35 and 25 OsSPL2,OsSPL14 and OsSPL17 interacted candidate genes were predicted by RiceNet,respectively.Moreover,they shared 20 common genes including six SPL genes,four zinc finger protein genes containing C2H2 and eight transcription factors.5.The expression of miR529a was induced by exogenous H2O2,which has the positive correlation to the concentration of exogenous H2O2.Overexpressing miR529a could increase seed germination rate,reduce leaf rolling rate,active root tip cell viability and increase chlorophyll retention rate,when subjected to exogenous H2O2 stress.Moreover,the expression of OsSPL2 and OsSPL14 was negatively correlated with the concentration of exogenous H2O2.The expression of ROS-responsive genes LOC_Os01g22249,LOC__Os05g38150 and LOC_Os04g59150 and H2O2-stress related genes Os10g0519700,OsVPE3 and CPR5 were all significantly up-regulated.These suggested that overexpressing miR529a could maintain the banlance of ROS metabolism to enhance the tolerance to exogenous H2O2 stress in rice.In summary,miR529a could regulate the expression of OsSPL2,OsSPL14 and OsSPL17 to control the growth and development of rice.This will provide a new ideas breeding of high yield rice varieties with the ideal plant architecture.On the other hand,miR529a can enhance the antioxidant ability of rice plants by negatively regulating the expression of target genes OsSPL2 and OsSPL14,it will provide the theoretical support for developing stress resistance rice.