Identification And Preliminary Study Of Rice MicroRNA Which Involved In Magnaporthe Oryzae Interaction

China is the world’s largest rice producer and consumer,with more than 60%of the population living on rice as the staple food.At the same time,as the world’s major food crop,rice production is directly related to food security in the world.Rice blast is one of the most serious diseases in rice production.The annual yield loss caused by rice blast is as high as 11%to 30%.In recent years,studies have found that micro RNAs participate in rice disease resistance pathways by regulating target genes,thereby increasing or decreasing rice blast resistance.Therefore,the identification and research of rice micro RNAs which related to rice blast can help us to deepen understanding of the mechanism of micro RNA in rice response to rice blast,and provide a theoretical basis for the control of rice blast.In this study,we used a stable high-resistance rice cultivar H4 and susceptible control line Zhong shuang zhan as materials,and validation of novel micro RNAs obtained by small RNA sequencing by Northern blot and stem-loop RT-q PCR.We have initially identified the target genes for novel micro RNAs by bioinformatics prediction and sequencing results of degradation groups.Then,we select two novel micro RNAs T21 and T34 subjected to preliminary functional studies.We obtained the following results:（1）The results of Northern blot and stem-loop RT-q PCR showed that 15 novel micro RNAs such as T15,T21,T22 and T52 were expressed in the disease-resistant and susceptible materials.The expression levels of T48,T49 and T50 were down-regulated within 0～72 hours of inoculation of resistant and susceptible materials,the expression levels of T15,T21 and T30 increased first and then decline;the expression levels of T25,T31,T34,and T42 were significantly different in the disease-resistant and susceptible materials.（2）We predicted a total of 2,260 target genes by 45 novel micro RNAs.And we found that the predicted target genes covered almost 175 genes obtained by sequencing in all degradation groups which containing 23 novel micro RNAs.It is indicated that the target gene prediction based on the principle of complementary pairing is more comprehensive.（3）Through bioinformatics analysis,we found that T21 and T34 were located on chromosomes 2 and 12 of rice.T21 and T34 are highly conserved in sequence and have high homology with micro RNA of lotus,soybean and human.The tissue-specific expression analysis indicated that T21 was mainly expressed in bud sheath and leaf,T34 was mainly expressed in stem,leaf and internode,and the promoters of T21 and T34 had obvious tissue specificity.（4）Transient expression of a GFP-target gene fusion expression vector system by tobacco,we verified that T21 may target polyamine oxidase gene Os PAO4 and cysteine synthase gene LOC＿Os05g51380.1;T34 may target protein phosphatase 2C family gene Os PP2C21 and protein Os FBX21 containing f-box domain.Subsequently,the inhibitory effects of T21 and T34 on the target gene were further confirmed by T21,T34 and target gene expression analysis and Western blot analysis.（5）Overexpression of T21 enhanced the sensitivity of rice to Magnaporthe oryzae,while Knockout and overexpression of target mimicry of T21 enhanced resistance to rice blast.We speculate that T21regulates the accumulation of H₂O₂ and affects the synthesis pathway of cysteine to negatively regulate rice blast resistance by inhibit Os PAO4 and LOC＿Os05g51380.1.Overexpression of T34 reduced the sensitivity of rice to Magnaporthe oryzae,while knockdown and overexpression of target mimicry of T34 reduced rice blast resistance,and the expression of T34 target genes Os PP2C21 and Os FBX21 was inhibited by Magnaporthe oryzae.It is speculated that T34 negatively regulates Os PP2C21 and expression of Os FBX21 regulates rice basal immune responses.Together,we identified 15 micro RNAs by Northern blot and stem-loop RT-q PCR,and preliminary confirmation that T21 negatively regulated rice blast resistance through target Os PAO4 and LOC＿Os05g51380.1,while T34 targeting Os PP2C21 and Os FBX21 increased the basic immune response of rice.We test and verify the sequencing results of previous small RNAs and provides a powerful basis for subsequent studies of these micro RNAs.At the same time,preliminary functional studies on T21 and T34 have deepened the understanding of micro RNA in rice response to the infection of rice blast fungus,and provided a theoretical basis for the prevention and control of rice blast.