Analysis Of Specific MicroRNA Gene From Tuber Of Dioscorea Alata

Dioscorea alata is rich in carbohydrates,protein and vitamins,and is an important organic food source in tropical and subtropical regions.The yield per mu of Dioscorea alata is high,but the shape of underground tuber is changeable.The research on the mechanism of the tuber swelling will lay a theoretical foundation for the cultivation of excellent Dioscorea alata varieties.Micrornas(miRNA)control plant activities,including plant growth,development,reproduction and stress reaction,potato miR172,miRNA 156 through participating photoperiod affect the tuber formation of potato crops,excessive expression of miR172 can accelerate the tuber formation under the condition of long sunshine,excessive expression miR156 can change shape and decline tuber yield.In this experiment using Dioscorea alata genome sequencing,spectrum and small RNA transcription data as the foundation,using bioinformatics tools seeking stems and tubers differentially expressed microRNAs,the screening of micrornas play a crucial role in the process of tuber formation,and predicted its target genes,then by RT-PCR,RT-qPCR analysis of micrornas and characteristics of the expression of target genes,and the application of fluorescence double reporting system verify the miRNA target genes,the following results were obtained:1,Through small RNA sequence analysis and comparison,139 known miRNA genes were obtained from Dioscorea alata,and small RNA data of common stem and tuber were compared and analyzed.44 miRNA genes with more than twice differential expression were obtained,including miR159,miR164,miR167 and miR168 families.The second-order structure of the precursor sequence of miRNA was predicted,and the length of the precursor sequence varied greatly.Within the 77-212 bp interval in the predicted mature miRNA sequence,the different gene loci of miR159 only had 1-2 base differences in the length of miRNA,and the 5 ends of the mature miRNA sequence were consisten.Target genes were predicted and the results were annotated.It was found that there were a total of 17 possible target genes in the 4 families.Different miRNA families might correspond to the same target gene,and the same miRNA family might correspond to different target genes.In order to further analyze which genes of the four miRNA families were expressed,we found the precursors of 13 mirnas based on the transcriptional spectrum data of Dioscorea alata.2,We used RT-PCR to detect which tissue did miRNA gene expressed among three types of tissues-leaf,stem and tuber.The results indicated that 5 miRNA genes from the four miRNA family were expressed in tuber,while the rest miRNAs were expressed in all detected tissues.3,To characterize the expression profiles of 14 mature miRNAs,which were identified from small RNA sequencing and differentially expressed between stem and tuber,RT-qPCR were carried out in 12 tissues,including leaf,stem and tuber tissues from tissue cultured seedlings and leaf,stem and tubers from four developing stages of field materials.The RT-qPCR showed that eight microRNAs from five miRNA family have higher expression quantity in tuber than that of other tissues.The eight miRNAs belongs to miR164(1),miR171(1),miR396(1)and miR168 family(5).While miR167,miR398 and miR156 had lower expression levels in tubers than that of other tissues.The rest of microRNAs had lower expression levels in leaf and stem tissues for plant grown in field.Majority results were correspongs to the miR-seq results.4,We used RT-qPCR to detect the expression profiled of 1 7 predicted miRNA targets in the set of samples same for miRNA expression detection.The RT-qPCR resuslts showed that most genes had lower expression levels for tubers tissues derived from field,except for DaLSH3-1、DaLSH3-2、DaAlfin,which had significant higher expression levels in tuber tissues.DaCO、DaGRAS1、DaTify、DaMYC showed higher expression levels in leaf than that of other tissues,while the rest genes had higher expression levels in stem than that of tubers.The above results showed that miR171-DaGRAS1/DaGRAS2,miR396-DaGRF1,miR159-DaMYC have negetive correlation.5,The vector for target gene insertion in the dual fluorescence reporting system were modified by adding 35S promoter and a multiclonal sites,and five miR156 gene sequences were added to provide materials for subsequent experiments.