| Water yam(Dioscorea alata)is an important staple crop and economic crop in the tropics,which has many merits,such as high yield,easy to cultivate and nourishing.However,the shape of water yam tubers is highly variable and irregular.At present,few researches have been conducted to reveal the molecular mechanism of how water yam tubers are formed and developed.MicroRNA(miRNA)in plants participates in the regulation of plant growth and development,as well as responses to environmental stress and other biological processes.Thus,analyzing the roles of miRNA on tuber formation and development in water yam is crital for improving tuber yield and breeding water yam cultivars with regular tuber shapes.In this study,we conducted systematic analysis of miRNA identification,target gene predication and annotation based on the genome,transcriptome and miRNA-seq datasets of water yam,explored their expression patterns via q RT-PCR,and screened key miRNAs and their target genes related to tuberization.Based on the above results,we selected miR156 and its target gene SPL(SQUAMOSA promoter-binding protein-like)gene family for further analysis.The exact cleavage site of DaSPL1 was detected by 5’-RLM-RACE.The interaction between DamiR156 and DaSPL was verified by fluorescence reporter system.The main results are as follows:1.The results of miRNAseq data showed that there were specially expressed miRNA members at different developmental stages and different tissues of water yam.Thirty miRNA members in the conserved miRNA families were selected,target genes of which are predicted and annotated.The expression patterns of miRNAs and target genes in plants with or withour tuberization were verified by q RT-PCR,and 22 miRNAs had significantly higher higher expression in tuber tissues than that of stem.Because the size of tubers were significantly different in pot-cultured and tissue-cultured plants,we compared the expression levels of miRNAs in tubers of pot-cultured and tissue-cultured plants,finding that there 13 miRNAs had significantly higher expression in pot-cultured plants than that of tissue-culture plants,including miR156-2,miR159-3,miR166-1,miR166-2,miR167-2,miR168-2,miR171-3,miR171-1,miR393-1,miR394-1,miR395-1,miR396-1,and miR535-1.These miRNAs may contribute to the phenotypic differences between the two types of water yam plants.Most miRNAs and their target genes have a negative relationship in their expressions.Based on the expression patterns of these miRNAs,we speculate that miR156,miR159,miR828,miR393,miR167,miR171 and miR168 may play a role in the development of tuber development.The selected miRNAs provide research targets in further research of tuber development.2.In this experiment,we predicted the miR156 precursors and identified eight miR156 gene loci,five of which could produce mature miR156 with complementary sequence sites of DaSPL genes.Among these miR156 loci,pri-miR156 a,b,e,g produce the same miR156 mature sequence,which has one nucleotide variation from the miR156 mature sequence produced by pri-miR156 c.The results of the sequence analysis of the DaSPL gene family showed that the four DaSPL transcripts contained the conserved target sequence of miR156,and the miR156 recognition sites of DaSPL1 and DaSPL2 were located in the 3’UTR region,and the miR156 recognition sites of DaSPL3 and DaSPL4 are in the CDS region.This results provides a reference for the functional verification of water yam miR156 and its target gene SPL gene.3.The 5’-RLM-RACE results showed the site where DaSPL1 is cleaved by miR156.One cleavage site is located in the middle of the target sequence,and the other two cleavage sites are located within 5 bases outside the target sequence.The fluorescence reporter experiment verified that the mature miR156 produced by Da-Pri-miR156 a and Da-Pri-miR156 c interacted with DaSPL.4.In this experiment,the DaSPL1 overexpression vector and DamiR156-STTM overexpression vector were constructed,and the concentration of hygromycin that caused100% death of the axillary buds of ’Dioscorea alata 40’ was 7.5 mg/L.This concentration can be used as the initial hygromycin concentration for subsequent selection of transgenic positive plants. |
