Identification And Analysis Of Related MiRNAs In Barley During Colchicine-Induced Doubling

Colchicine is a common reagent for doubling plant chromosomes and artificially polyploids.It is a kind of toxic alkaloid.When treated with colchicine solution,plants will react to the potential stress of water and colchicine.However,little is known about the role of microRNA in response to colchine in plants.Here,we used cultivated barley was used as a model plant,and set up no treatment group,5h water treatment group,and 5h colchicine treatment group.TheRNAs from treated root samples were uesd to build 3 smallRNA libraries for mining differentially expressed miRNAs in response to colchicine treatment,and exploring the molecular regulation mechanism of miRNAs in plants during colchicine treatment.1、High-throughput sequencing was performed on these three constructed smallRNA libraries,and a total of 54 conserved barley miRNAs and 76 novel barley miRNAs were found from these three libraries.The conserved miRNAs belonged to12 miRNA families.Most of the smallRNAs in these three libraries are 18 nt and19nt,followed by 20 nt and 16 nt.2、Differential expression analysis showed that there were 39 and 42 differentially expressed miRNAs in HV5 CK vs HV0 and HV5 TR vs HV0,respectively.Compared with HV0,18 and 24 novel miRNAs in HV5 CK and HV5 TR were up-regulated,and two identical conserved miRNAs were up-regulated.While there were 16 and 13 novel miRNAs and three identical conserved miRNAs down-regulated in HV5 CK and HV5 TR.Nine differentially expressed novel miRNAs were identified between HV5 TR vs HV5 CK.Compared with HV5 CK,there were 5 novel miRNAs upregulated and 4 miRNAs down-regulated in HV5 TR.3、GO and KEGG enrichment analysis of differentially expressed miRNAs in HV5 CK vs HV0 and HV5 TR vs HV0 showed that most of the high-degreeenrichment was concentrated on the functions related to the regulation of cell wall and cellulose anabolism.4、GO and KEGG enrichment analysis of the targets functions of 9 different miRNAs in HV5 TR vs HV5 CK showed that novel＿mir69,novel＿mir56,and novel＿mir25 jointly target the genes involving DNA repair pathway.The remaining targets are involved in processes related to cell division.5、qRT-PCR analysis was performed on 8 differentially expressed novel miRNAs in HV5 TR vs HV5 CK.The expression trends of 7 novel miRNAs were consistent with the sequencing data.The expression level of one novel miRNA(novel＿mir38)was slightly different.PI 11z37 was treated with water and colchicine for 24 hours.In PI 11z37,only the result of novel＿mir69 was opposite to 5h,and the expression of the remaining miRNAs was consistent with 5h.6、protein-protein interaction analysis results showed that the target genes MLOC＿68431.4 and MLOC＿66568.3 of hvu-mi R6214 were the highest scored interaction protein genes.The interaction protein genes with higher scores in the other two groups were MLOC＿81801.1 and MLOC＿54574.2 and MLOC＿54574.2 and MLOC＿12066.3.The results showed that these genes may be related to cellulose and methylation.While the target genes of 8 novel miRNAs in HV5 TR VS HV5 CK form a large number of interacting protein gene networks.7、Thirty-three target genes,with the function of DNA repair,jointly targeted by novel＿mir69,novel＿mir56,and novel＿mir25 in GO enrichment analysis were examined.An inverse relationship was observed between two miRNAs(novel＿mir69and novel＿mir56)and their predicted target genes,and another novel miRNA(novel＿mir25)did not show an inverse relationship with its predicted target genes.Since the regulatory mechanism between miRNA and its target genes is very complex,one miRNA may correspond to multiple target genes.