Mining And Functional Verification Of Stripe Disease Resistance Gene Of Hulless Barley Based On MiRNA

Micro RNA（miRNA）is a class of non-coding small RNA of 19 ～ 24 nt generated by a single-stranded RNA precursor with hairpin structure.It plays an important role in plant growth,development and stress response.At present,there are many studies on plant and pest-related miRNA,especially in Arabidopsis thaliana,Brachypodium distachyon,tobacco,rice,maize,barley and other crops.However,the study of miRNA related to hulless barley and it’s diseases has not been reported at present.Among all the diseases and insect pests of hulless barley,stripe disease is one of the main fungal diseases that reduce the yield of hulless barley heavily.In this study,through the combination of miRNA database construction and functional verification experiment,we discovered,predicted and identified the miRNA,closely related to stripe disease in hulless barley,and analyzed the related functions of these miRNA.It was revealed that hvu-mi R168-5p and its predictive target gene hvu-AGO1 were related to the resistance to stripe disease in hulless barley.The results of high-throughput sequencing showed the expression level of hvu-mi R168-5p increased in resistant cultivar "Kunlun 14" and susceptible cultivar "1141" of hulless barley after they were infected with stripe disease.In this study,we cloned the hvu-AGO1,which is predicted as the target gene of hvu-mi R168-5p,and found that the expression of hvu-AGO1 gene decreased after the infection of stripe disease.The results of semi-quantitative PCR confirmed a negative correlation between the expression of hvu-mi R168-5p and hvu-AGO1 gene,which was in accordance with the regulation pattern of miRNA and target gene.The results not only deepen the understanding of the resistance mechanism of hulless barley to the stripe disease,but also lay a foundation for the breeding of hulless barley resistance to the stripe disease.The main results in this study are as follows:（1）Construction of infecting system of hulless barley with the stripe disease in laboratory:（1）the infected leaves of resistant strains "Kunlun 14","Kunlun 15" and susceptible strains "1141" and "1140" of hulless barley were collected.3-4 pieces（5 ×5 mm）of the treated infected leaves（2）were cultivated in PDA plate for 7 days.The fungal cake was picked out with a perforator and cultivated in a new PDA plate,with the temperature at 22-25 ℃.The whole plate was filled with the stripe disease colony after 6 days or 8 days of cultivation.（3）The seeds of hulless barley were washed with75% alcohol and placed in a plate with the stripe disease colony.The seeds were cultivated at low temperature of 6 ℃ for 15 days.（4）The treated seeds were planted in a flowerpot with vegetative soil and cultured in an artificial climate incubator（14 h,20 ℃ in the day,10 h,10 ℃ in the night）.After 10 days,the fungal hyphae of the stripe disease could be observed.The complete process took about 40 days or 45 days.（2）Screening for resistant miRNA of hulless barley against the stripe disease: 92 miRNAs of hulless barley were found before and after the infection of "Kunlun 14" and "1141" strains by means of miRNA-seq miRNA database construction and analysis.Among them,36 of miRNAs were reported,as well as 56 of which were newly discovered miRNAs.We discovered 35 miRNAs with significant expression difference before and after infection of hulless barley stripe disease.By comparing the difference of these miRNAs in resistant variety（Kunlun 14）and susceptible variety（1141）,four critical miRNAs are found,two of them are known miRNA（hvu-mi R168-5p and hvu-mi R6198）and the other two of unknown miRNA（novel-11 and novel-91）.The statistical software Stst2 and SPSS13.0 were used for analysis of variance and multiple comparison.It was found that the expression of them was significantly different before and after infection（P < 0.01）.（3）Hvu-mi R168-5p target gene prediction analysis: using plant miRNA target gene on-line prediction software ps RNATarget,hvu-AGO1 was discovered one of the target genes of hvu-mi R168-5p.Through biological information software analysis and literature reference data mining,hvu-AGO1 involved in plant development and stress response.It is suggested that hvu-mi R168-5p may participate the resistance progress of hulless barley to stripe disease by regulating the expression of hvu-AGO1 as the target gene.（4）Relationship between candidate miRNA and its target gene: the target gene hvu-AGO1,was cloned by RT-PCR,combined with RACE technique to obtain the complete CDS of the target gene hvu-AGO1,as well as the homologous 5’RACE fragment.High-throughput sequencing showed the expression level of hvu-mi R168-5p increased after infection with stripe disease in the resistant variety "Kunlun 14" and susceptible cultivar "1141" of hulless barley while the expression of target gene hvu-AGO1 decreased by semi-quantitative PCR detection.Therefore,the predicted hvu-mi R168-5p was negatively correlated with the target gene hvu-AGO1 at the level of gene expression.