Identification And Expression Analysis Of MicroRNAs In Drought-resistant Wild Soybean (Glycine Soja)

Small RNA (MicroRNA, miRNA) plays important roles in post-transcriptional gene silence bydirecting target mRNAcleavage or translational inhibition. Wild soybean (Glycine soja) is considered tobe the most important gene sources for genetic modification of domesticated soybean. Identification andfunctional analysis of miRNAin the Glycine soja will further promote the utilization of genetic resoursesfor soybean breeding. This study used two approaches to separate and identify miRNAin accession S101of drought-resistant wild soybean: concatenating cDNA fragments for sequencing and high throughputsequencing. Combining with examinations of putative miRNA-directed cleavage products, constructingartifical pre-miRNAs and transformation of Arabidopsis thaliana, we performed preliminary functionalresearch for novel miRNAs.This study obtained important information from separation and identification of miRNAin Glycinesoja, and broadened the current understanding and awareness of plant miRNAs. The detailed results areas follws:1.Data analysis of small RNAlibraryfrom concatenation forsequencingThrough constructing a small RNA library and sequencing after concatenation, we obtained 2,880high quality small RNAsequences. The distribution map indicated that 1,347 (1,022 Unique) were 19-24nt in length, which composing 46.8%. The most abundant class was 21 nt, which contained 334sequences (223 unique).①Comparing these small RNAs with the mature miRBase (release 11.0) by BLASTN, we found15 conserved miRNAs homologs (≤2bp mismatches), in which 14 were conserved with domesticatedsoybean. The 15 conserved miRNA candidates can compose 8 families: miR156, miR159, miR160,miR167, miR168, miR171, miR319 and miR396. Except miR171, all these conserved families had beenpreviouslypredicted in domesticated soybean.②Analysed combining the domesticated soybean EST database, 9 of the 15 homologs could findperfectly matched hits on 27 ESTs. 17 ESTs were consistent with the reported soybean miRNAprecursors except 10 ESTs (corresponding to miR167 homolog), which had been classified as novel twoputative gso-miR167 precursors after secondarystructure analysis.③For novel miRNAprediction, 74 small RNAin 442 candidates could find perfect matching hitsin 407 soybean EST sequences. As a result, 22 ESTs corresponding to 9 small RNAs (8 families) wereidentified with characteristic hairpin structures, which were deemed as novel miRNAcandidates.④All these miRNA candidates (15 conserved and 9 novel) were 20~22 nt in length and most oftheir 5'terminal nucleotides were uridine, which was consistent with the miRNAcharacteristic.2.Data analysis of small RNAlibraryfrom high throughput sequencingThe next-generation sequencing technology (Solexa) afforded an effective approach foridentification of miRNAs. Using the Solexa sequencing technology, we constructed a small RNAlibraryand obtained 3,161,992 high quality reads (1,282,308 Unique).①Combining the recent soybean genome database (Glyma1), bioinformatics analyses indicated that 12,734 miRNAcandidates passed RNAfold and MIRcheck evaluation, which shown the complexityof miRNA groups. Finally, 141 conserved miRNAs comprising 34 families and 171 novel miRNAscomprising 100 families were identified in wild soybean after strict screening and manual conformed.②Among 171 novel miRNA candidates, 206 putative target genes with various functions werepredicted for 53 miRNAs, which were involved in varied aspects of environmental responses anddevelopmental processes in plants.③Additionally, based on the high through-put advantage, some interesting phenomena wereobserved from further analysis. Such as: tandem patterns of miRNA in miR159 and miR319 precursors,some miRNA* accumulation, and querying the qualification of some gam-miRNAs which seemed to begenerated fromdouble strands.3. Preliminary functional analysis of miRNAsWe performed preliminary functional analysis of 8 conserved and 8 novel miRNAfamilies obtainedfrom small RNA library. Such as, Nothern blot,target prediction and transformation of Arabidopsisthaliana.①Northern blot analysis indicated that all the 8 conserved and 8 novel miRNA families weredetectable in wild soybean seedlings under normal or drought stressed conditions. Among them, fourconserved and three novel miRNAs displayed different abundance in respond to drought stress. miR160,miR167,miR319,miR396 and gso-miR2 were down-regulated, while gso-miR1 and gso-miR6 wereup-regulated. Aiming at the 8 novel miRNA families, Northern blot analysis were also performed in inroots, stems and leaves of wild soybean. Most of them were inclined to express in roots and exhibitedhigh tissue-specific expression, which suggested that the miRNAs'biogenesis and functions might bevaried in different organs.②Target prediction found 32 genes as putative targets for 7 novel miRNAs. Except that part ofthese targets was not annotated, most of them were resistant proteins. We examined the presence ofputative miRNA-directed cleavage products by 5'-RACE experiments. Two of these potential targetswere validated, TC233731 for gso-miR7 and TC225607 for gso-miR8. Targets of gso-miR8 were afamilyof resistant proteins in whichTC225607 coding for an R 9 protein.③Through constructing miRNAexpression vector and transformation of Arabidopsis thaliana, thepreliminary functional research (gso-miR5,gso-miR6,gso-miR7 and gso-miR8) indicated that mosttransgenic plantlets of gso-miR8 exhibited earlyflowering mutant phenotype.