Identification And Functional Characterization Of Drought-resistance Related MicroRNAs And Their Targets In Mulberry

MicroRNAs?miRNAs?are endogenous non-coding small RNAs which are approximately 21 to 24 nucleotides single-stranded and regulate gene expression accurately and effectively at post-transcription level by means of the negative regulation of target mRNA expression on degradation or translational inhibition.Plant miRNAs can response abiotic stress,such as drought,NaCl,low temperature,and play an important function in the course of stand up to adversity.Various adversity conditions have a significant impact on the growth of mulberry?Morus alba L.?.The research of the function of the miRNAs and their target genes,and the physiological and biochemical and molecular mechanism of responding to stresses in mulberry can play important roles on improving the resistance of mulberry,and preserving mulberry germplasm resources and increasing production of mulberry.At the same time,this can provide scientific support for the development and utilization of mulberry as ecological forest tree species.In this thesis,drought-resistance related miRNAs and target mRNAs were identified via the high throughput degradome sequencing.The drought-resistance functions of candidate miRNAs and target mRNAs were analyzed by transient transformation and virus induced gene silencing?VIGS?,and the molecular mechanism of drought resistance in mulberry was explored.The results of the study are mainly as follows.1.High throughput deep degradome sequencing reveals microRNAs and their targets in response to drought stress in mulberryIn control library?CL?,409 target genes of 30 conserved miRNA families and 990 target genes of 199 novel miRNAs were identified.In drought library?DL?,373 target genes of 30conserved miRNA families and 950 target genes of 195 novel miRNAs were identified through high throughput deep degradome sequencing.Of the conserved miRNA families in DL,miR156,miR172,and miR396 had the highest number of targets,indicating that these three miRNA families and their target genes might play important functions in response to drought stress in mulberry.By analyzing the miRNA-target molecular network,we found that the DL independent networks consisted of 838 miRNA-mRNA pairs?63.34%?.The expression patterns of 11 target genes and 12 correspondent miRNAs were detected using qRT-PCR.Six miRNA targets were further verified by RNA ligase-mediated 5'rapid amplification of cDNA ends?RLM-5'RACE?.Gene Ontology?GO?annotations and Kyoto Encyclopedia of Genes and Genomes?KEGG?pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways.2.Characterization and functional analysis of miR166f in drought stress toleranceThe precursor of miR166f was isolated,cloned,and identified from the leaves of mulberry,and the pre-miR166f was 91 bp in length with a minimum folding free energy of-51.70 kcal/mol.Bioinformatics analysis showed that the 3000-bp 5?upstream region of miR166f contained not only TATA and CAAT boxes that functioned in transcription initiation,but also had many cis-acting elements for stress responses and several hormone response elements.We established a transient transformation system in mulberry.Histochemical?-glucuronidase?GUS?staining of miR166f-overexpression in transient transgenic mulberry leaves showed the best effect and highest GUS gene expression compared to the wild-type control plants at day four post-transformation using an optimized concentration of transformation liquid(OD₆₀₀=0.7).The target genes of miR166f?two HD-Zips and one histone arginine demethylase gene?showed markedly lower expression levels in miR166f-overexpression transient transgenic mulberry leaves.Further investigation showed that transient transgenic mulberry trees had higher relative water content,free proline content,soluble protein content,and superoxide dismutase and peroxidase activities,and lower malondialdehyde content compared to the wild-type control plants.The results suggested that miR166f might function as a positive regulator of drought stress tolerance in mulberry.3.Characterization and expression patterns of shabby-related kinase?MmSK?gene and its prokaryotic expression in E.coliA cDNA sequence encoding MmSK?GenBank accession NO:KY348867?was cloned from the leaves of mulberry,which was 1705 bp in length with a full open reading frame?ORF?of 1236 bp encoding a protein of 411 amino acids.The estimated molecular weight and isoelectric point?pI?of the putative protein were 46.55 kDa and 8.61,respectively.Conservation domain structure analysis indicated that MmSK protein had typical structure of the protein kinase domain and belonged to GSK3/shaggy protein kinase family.Multiple sequence alignment and phylogenetic analysis showed that the homology between the amino acid sequences encoded by the MmSK gene and various species was more than 89%.Quantitative real-time PCR?qRT-PCR?analysis revealed that MmSK was expressed in all the tested tissues with the highest expression in the phloem.The expression level of the mRNA has changed significantly under salt,drought,cold and ABA stress treatments compared to the normal growth environment.The ORF segment of the MmSK was inserted into the expression plasmid pET-28a?+?to construct a recombinant expression plasmid.SDS-PAGE and western blot results revealed that His-tagged fusion protein was successfully expressed.4.Establishing of the VIGS system and function identification of MmSK in drought stress toleranceUsing phytoene desaturase?PDS?gene as report gene,the VIGS system in mulberry was successfully constructed.Mulberry MmSK gene was successfully silenced by VIGS,and after MmSK was silenced,the expression of MmSK in pTRV2-MmSK-VIGS plant dropped to 34.02%compared with the negative control inoculated with empty vector pTRV2-00.Under drought stress,the soluble protein content,proline content,superoxide dismutase?SOD?and peroxidase?POD?activities in mulberry seedlings with silenced MmSK gene decreased in different degree compared with the pTRV2-00 plant.In contrast,the malondialdehyde?MDA?content increased significantly.With the extension of drought stress treatment time,the soluble protein content,proline content and MDA content gradually increased.The SOD activity and POD activity under drought stress gradually rose to the maximum on the fifth day and then decreased,which consistent with the change trend of MmSK gene expression.In a word,the physiological and biochemical characterization suggest that the drought stress tolerance of MmSK-suppress-expression transgenic mulberry was weakened considerably compared to the control plants and MmSK could function as a positive regulator of drought stress in mulberry.In this study,drought-resistance related miRNAs and target mRNAs were identified via the high throughput degradome sequencing and the time-space characteristics of candidate miRNAs and their target mRNAs were confirmed.Moreover,the influence of miR166f on expression of target genes and drought-resistance related physiological and biochemical indexes of plant were analyzed by overexpression.VIGS transformation system was established in mulberry,and the drought-resistance function of MmSK gene was evaluated by VIGS.The results helped to improve the molecular regulatory network of drought resistance in mulberry and provided important avenues of exploration and theoretical base for mechanism of resistance to drought and resistance breeding of mulberry.