| Sunflower rust caused by Puccinia helianthi Schw is one of the most important fungal diseases on sunflower,which seriously affect the quality and oil content.MicroRNA(miRNA)is a kind of non-coding single-stranded small RNA that is commonly found in plants,and is involved in regulating plant growth and development and various stress responses.Aiming at the research gap of sunflower resistance-related miRNAs,small RNA library of sunflowers under rust disease stress was constructed in this experiment,and miRNAs significantly related to disease resistance were selected by Solexa high-throughput sequencing and bioinformatics analysis.qRT-PCR technology was used to quantitatively verify ten disease-resistant miRNAs,and target genes were predicted using miRanda software;four disease-resistant miRNAs were selected to construct miRNA over expression vectors.This lays the foundation for further research on the molecular mechanism of sunflower miRNA resistance and further research on the function of sunflower-related miRNAs.The main findings are as follows:1.Using high-throughput sequencing technology to predict and analyze sunflower related miRNAs,a total of 378 miRNAs were found in its entire genome,of which 373 were newly discovered miRNAs,and 13 of the newly predicted miRNAs were the control group(A),the disease resistance group(B),and the susceptible group(C).There are eight differentially expressed genes between sample A and sample B,three differentially expressed genes between sample B and sample C,and two differentially expressed genes between sample C and sample A.2.Ten differentially expressed miRNAs(Han-miRll,Han-miR21,Han-miR22,Han-miR25,Han-miR34,Han-miR42,Han-miR43,Han-miR45,Han-miR47,Han-miR67)were selected for qRT-PCR verification(Screen out the two differentially expressed miRNA genes in A-VS-C,and consider that there is little relavance to the study of disease-resistant miRNA in this experiment;screen out another differentially expressed miRNA because its corresponding target genes are less).The detection results of qRT-PCR were consistent with 80%of the previous high-throughput sequencing results.3.The miRanda software(3.3a)was used to predict target gene and annotate 13 differentially expressed miRNAs.A total of 155 target genes are predicted for the 13 differentially expressed miRNAs,each miRNA corresponds to multiple target genes.A small number of target genes have unknown functions,122 target genes obtained their functional annotation.4.Functional classification of 13 differentially expressed miRNA target genes(128 in total)using GO,the results showed:"ADP binding activity"and "Purine nucleoside binding activity","Nucleoside binding activity","Purine riboside""Purine ribonucleotide binding,ribonucleoside binding,purine ribonucleoside binding and purine nucleotide binding" are GO terms that are significantly enriched in molecular functions;"Response to stress,defense response" are GO terms that are significantly enriched in biological processes.5.The target genes of 13 differentially expressed miRNAs were annotated with KEGG pathway and analyzed by significant enrichment,the results showed that sample A and B(B-VS-A),sample B and C(B-VS-C),sample C and A(C-VS-A),which respectively involved in five types of biochemical metabolic pathways in material metabolism,organic systems,genetic information processing,environmental information processing,and cytological processes.In B-VS-A,the target genes of differentially expressed genes have 38 annotations to 33 pathways,and in B-VS-C,the target genes of differentially expressed genes have 26 annotations to 23 pathways,in C-VS-A the target genes of differentially expressed genes have seven annotations to five pathways.Caffeine metabolism,Bacterial secretion system,Plant-pathogen interaction,Gap junction,Quorum sensing are significantly enriched pathways,which clearly shows that the enriched pathways are mostly related to the biosynthesis of materials,the biological metabolism of cells,and the process of disease stress.6.Compared with control group A,four target genes of Han-miR21 with up-regulated expression and five target genes of Han-miR43 with down-regulated expression in resistant group B were selected to further tested.It was detected that one target gene of Han-miR21 was down-regulated and three target genes of Han-miR43 were up-regulated,which were negatively correlated with the expression of Han-miR21 up-regulated and Han-miR43 down-regulated,which is in line with the regulation of miRNA and its target genes.7.Four miRNAs of sunflower expressing vectors(pBI121-Han-miR21,pBI121-Han-miR25,pBI121-Han-miR43,pBI121-Han-miR45)were successfully constructed. |
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