Identification And Interaction Of Grapevine MicroRNA164 And MicroRNA171 And Their Targets

Grapevine(Vitis vinifera L.)is an important fruit trees in the world and play an important role in the agricultural economy of China,the yield of grapevine is second to Citrus.Grapevine has finished the whole genome sequencing project which to be ranking first among all the fruit trees.miRNAs and transcription factors plays a crucial role in a variety of biological and metabolic pathways in plant,such as leaf morphogenesis and differentiation,identification and differentiation of floral organs,organ development,signaling pathways and biotic and abiotic stress responses.The table grapevine ’Summer black’ and ’Fujiminori’ were used as experimental materials in this study where identification and characterization of NAC,GRAS transcription factors family,miR164/171 and its target genes and identification of grape bud miRNAs during the dormancy by bioninformatics tools,qRT-PCR,PPM-RACE,RLM-RACE,transgenic and high-throughput sequencing.The major works and findings were as follows:1.To investigate the extent and degree of evolutionary conservation of miR164 in Arabidopsis,grape,poplar and rice,apple,maize,the phylogenetic relationships among miR164 precursor and mature sequences was reconstructed.Despite strong conservation,some mature miR164 sequences,such as osa-miR164c,have undergone critical sequence variation,leading to functional diversification,since they target non-NAC gene transcript(s).Phylogenetic analyses revealed a combination of old ancestral relationships and recent lineage-specific diversification of the miR164 family within four model plants.The cis-regulatory motifs on the upstream promoter sequences of miR164 genes were highly divergent and shared some similar elements,indicating their possible contribution to the expression patterns and functions diversified.Reconstructed the phylogenetic relationships among miR171 precursor and mature sequences so as to investigate the extent and degree of evolutionary conservation of miR171 in Arabidopsis thaliana(L.)Heynh.(ath),grape(Vitis vinifera L.)(vvi),poplar(Populus trichocarpa Torr.&A.Gray ex Hook.)(ptc),and rice(Oryza sativa L.)(osa).Despite strong conservation of over 80%,some mature miR171 sequences,such as ptc-miR171j-1,and-m and osa-miR171g,-h,and-i,have undergone critical sequence variation,leading to functional diversification,since they target non-SCL gene transcript(s).Phylogenetic analyses revealed a combination of old ancestral relationships and recent lineage-specific diversification in the miR171 family within the four model plants.The cis-regulatory motifs on the upstream promoter sequences of miR171 genes were highly divergent and shared some similar elements,indicating their possible contribution to the functional variation observed within the miR171 family.2.By utilizing the bioinformatics method,we analyzed the phylogenetic of 143 NAC family protein sequence and NAC genome localization firstly,and then predicted and analyzed their amino acid composition,physical and chemical characteristics,as well as secondary and tertiary structures,meanwhile we still assayed the relation of NAC gene family between grapevine and Arabidopsis thaliana.The result showed that these 143 amino acid sequences could be clustered into sixty groups together with 94 NAC sequences of Arabidopsis thaliana,which illuminated that NAC genes had highly conservative between grapevine and Arabidopsis thaliana.Further genetic mapping of NAC genome localization found that they could be distributed on 17 chromosomes.The research still found that the number of amino acid and hydrophobic of amino acid sequences in different subfamilies presented some differences,however,the results of secondary structure predicted that the main composition of 143 amino acid sequences was random curl and the tertiary structure of all the sequences were quite similar.43 GRAS genes were identified from 12×ssembled grapevine genomic sequences.The duplication patterns,genomic structures and phylogeny of these 43 grapevine GRAS genes were investigated.To understand the roles of VvGRAS during grapevine development,their expression profiles in different tissues including leaf,tendril,inflorescence,stem,root and berry were tested using quantitative real-time PCR.Analysis revealed expression diversity of various VvGRAS genes among different grapevine tissues.To identify candidate grapevine GRAS genes with a role in response to stress,publicly available microarray data were obtained to calculate their expression change under abiotic and biotic treatments,with a number of VvGRAS genes displaying up-regulation and down-regulation after stress induction.3.The expression of VvmiR164 and VvmiR171 was detected in grapevine leaves,stems,tendrils,inflorescences,flowers and fruits by qRT-PCR.In addition,two potential target genes for VvmiR164/miR171 were also found and verified by PPM-RACE and RLM-RACE.The results not only maps the cleavage site of the target mRNA but allowed for detection the expression pattern of cleaved fragments that can indicate the regulatory function of this miRNA on its target genes.These target genes were explored by qRT-PCR where some exhibited different expression patterns from their corresponding miRNA,indicating the cleavage mode of the miRNA on its target genes.Transgenic Arabidopsis plants over expression VvSCL15 showed decrease tolerance to drought and salt treatments.4.The ’Fujiminori’ was used as material,and then two miRNA libraries and were conducted from top bud and bottom bud,miR164d and miR171 play important roles in bud development,and 79 conserved miRNAs belonging to 46 miRNA families were identified in the two libraries.Additionally,248 novel miRNAs were also detected.Of all these miRNAs,11 miRNA members were up-regulated,whereas 77 miRNAs were down-regulated in top bud.