Identification And Functional Analysis Of Drought-related MicroRNAs In Potato

Potato(Solanum tuberosum L.) is one of the most important crops in the world.But the growth and productivity are severely affected by drought stress. Plants respond to drought with an array of biochemical and physiological adaptations mainly depend on regulate expression of related genes. Until now, a large number of genes that respond to drought stress at the transcriptional level have been identified.However, detailed regulation mechanisms of those genes are incompletely understood.Micro RNAs(mi RNAs) are classes of small single-stranded non-coding RNAs, which can play important roles in the regulation of gene expression at the post-transcriptional level and have been considered to mediate gene silencing in plant development and response to abiotic stress. There have been increasing investigations on discovering mi RNAs and analyzing their functions in model plants. Up to now, little is known about mi RNAs and targets related to drought that have been identified in potato.In this reasch, We used bioinformatics identified three novel mi R159 and four novel mi R169 family members, Also identified mi RNAs which regulate proline enrichment under drought stress in potato; We also used micro RNAs sequencing and degradome sequencing to global survey drought-related mi RNAs and their targets in potato. and a large number of drought-related mi RNAs and potentially target genes were obtained, also some of drought-related mi RNA targets were validated by5’-RLM-RACE assay. The results are as follows:1. Based on mi R159 and mi R169 repressed the R2R3 MYB and NF-YA TFs are conserved in plants. Three potato mi R159 family members and four potato novel mi R169 family members were identified by bioinformatics analysis. Target prediction showed that mi R159 may regulate GAMyb-like TFs and mi R169 may regulate NF-YA TFs. Also those target genes have been cloned in potato, We further measured expressional levels of identified mi RNA and potential target genes during the different periods of drought treated samples using quantitative real-time PCR(q RT-PCR). The result showed that identified mi RNAs and some of their target genes had a negative expression pattern. So, we suggested that the mode of mi R159/169 regulate MYB and NF-YA TFs are conservative in potato.2. In order to understand whether mi RNAs related to regulate proline enrichmentunder drought stress, We used m RNAs of related genes as the targets of mi RNAs to search mi RBase and find 11 known mi RNAs classified into 6 mi RNA families as query mi RNA sequences. After q RT-PCR assay and functional analysis, we identified mi R172, mi R396 c, mi R396 a and mi R4233 may regulate P5 CS gene, and mi R2673 and mi R6461 may regulate P5 CR and Pro DH gene, respectively. The findings can help us make a good understand of the roles of mi RNAs in regulation of proline accumulation under drought stress.3. To global investigate potato mi RNAs involved in the response to drought stress,High-throughput sequencing was used to survey drought-related mi RNAs from four small RNA libraries of drought-tolerant and drought-sensitive potato genotypes both control and drought-stressed conditions. Based on sequence similarity and hairpin structure prediction, a total of 1,338 potato mi RNAs were identified, which included59 previously known mi RNA and 1,279 novel candidate mi RNAs in potato.Differential expression analysis showed that 318 mi RNAs were differentially expressed in potato cultivar Atlantic and 89 mi RNAs were differentially expressed in potato cultivar Zihuabai. And target genes of those differentially expressed mi RNAs were also predicted.4.To identificate the function of those differentially expressed mi RNAs,Degradome sequencing was used to identificate the mi RNA targets, and 90 mi RNA cleavage sites of 84 target genes were identified from a mixture degradome library.Based on targets annotation, 12 target genes were identified are drought related genes,and 7 of 12 were validated by 5’-RLM-RACE assay. which involved in drought-related functional protein(ascorbate peroxidase, glutathione S-transferase and ubiquitin-conjugating enzyme), protain kiase(serine/threonine kinase) and transcription factors(HD-Zip, SPL, NAC and Zinc-finger protein). The mi RNAs high-throughput sequencing and q RT-PCR expressional analysis showed that the expression of those mi RNAs were differential within drought-tolerant genotype“Zihuabai” and drought-sensitive genotype “Atlantic”. The results might provide some useful insights that mi RNAs may play important roles in drought tolerance in potato.