Identification And Spatiotemporal Expression Characteristics Of MicroRNAs And TasiRNAs In Different Organs And Development Stages Of Wheat(Triticum Aestivum.l)

Wheat is one of the most important food crops. Wheat production, yield and quality arerelated to world food safety. Formation and development of wheat seeds have direct influenceon wheat yield and quality. MicroRNAs（miRNAs） belonging to noncoding small RNAswere recently found universally existing in eucaryon with important gene regulation function.It ranges from20to25nucleotides in size. Many studies found that miRNA involved inpost-transcriptional regulation of gene expression. It also has important regulation function inplant growth, development, stress response and other biological process. By far, plantmiRNA research has made great progress. Totally10892plant mature miRNAs have beenregistered in the miRBase （Release21, June2014; http://www.mirbase.org）. In which mostof the miRNAs were from plant species with known genome sequence, such as rice （713）,poplars （401）, Arabidopsis （427）, Brachypodium distachyon （525） and corn （321）.Regulation functions of miRNAs in plant seed development have been studied in rice,Arabidopsis, corn, barley, and so on. By contrast, wheat has complex genome and largegenome size, wheat genome sequence assembling need to be accomplished. Few wheatsmiRNA have been identified. Only119miRNAs have been registered in the miRBase.Therefore, identification and functional analysis of miRNAs in wheat need to bestrengthened.Chinese wheat cultivar Xiaoyan6with high quality was used as material in this study.Wheat seedlings, flag leaves and immature seeds of5days,10days and20days afterflowering were collected for sRNA libraries construction and deep sequencing. BothmiRNAs and trans-acting siRNA （tasiRNAs） were identified from5sRNA libraries.Spatiotemporal expression patterns of these identified miRNA in different organs anddifferent development stage were studied. Target genes of these identified miRNA werepredicted to underlying their function primarily. In addition, possibilities of using BarleyStripe Mosaic Virus （BSMV） vector for overexpression of artificial miRNA or miRNA inwheat were studied. The objective is of this study to identify miRNAs specially expressed in flag leaves and different seed development stages, rich wheat miRNA information inmiRBase, and provide fundamental basis for further study on miRNA functions in wheatgrowth, development, especially regulation functions and network of flag leaves and seedsgrowth and development.The main research contents and results of this study are as follows:1. wheat (Triticum. aestivum L.) cultivar Xiaoyan6was used in this experiment. Wheatplants were grown under natural conditions, Wheat seedlings (at five-leaf stagepost-vernalisation), flag leaves of heading plants and immature seeds at5days-post-anthesis(5-d seed),10days-post-anthesis (10-d seed) and20days-post-anthesis (20-d seed) werecollected, and the total RNA was extracted, respectively. Five sRNA libraries wereconstructed and analyzed by high-throughput deep sequencing.13931738,13595341,14569411,14184881, and18311762clean reads of sRNA sequences was acquiredcorresponding to seedlings, flag leaves,5-d seeds,10-d seed and20-seeds, respectively. Thesequencing data were deposited in NCBI Gene Expression Omnibus (GEO,http://www.ncbi.nlm.gov.geo/) under the accession number GSE50524. The length distributionanalysis on sRNA reads obtained from the five sRNA libraries indicated that75%sRNA inthe seedling distributed in the range of20-24nt; sRNA reads from flag leaves werecharacterised with24,21and20nt (27%,30%and22%, respectively) as the major sizeclasses, The sRNA reads from the5-d,10-d and20-d seeds shared similar size distributionpatterns, with24nt as the dominant class of sRNAs（above50%）.Unique sequences areconsidered,24nt reads consist the predominant class of sRNAs (approximately55%to70%)in all the five libraries or tissues, as compared with the20to23nt reads (3%to15%). Theseresults indicate that the majority of sRNAs in the different organs of wheat are associatedwith RNA-dependent DNA methylation and hereochromatinic regions mediated genesilencing. In addition, The five sRNA libraries shared less than1.2%of the21nt sRNAs andless than0.4%of the24nt sRNAs, indicating diverse sRNA populations in the tested tissues.These results indicate that distinct pools of sRNAs work in different wheat tissues ordevelopmental stages Sequence between unique sRNAs and wheat genome sequence or ESTsequence were aligned, and the results showed that55%sRNA could be matched perfectly tothe wheat genomic shotgun assembly sequence, and28%sRNA matching perfectly to wheatEST sequence. All the identified sRNA has been clssified and annotated; the known miRNA(non-wheat miRNA from miRBase) and non-annotated sRNA sequence will be used as thedata source for the subsequent identification of known and novel wheat miRNAs.2. The wheat miRNA were identified by bioinformatic method from the known miRNA(non-registered miRNA in miRBase/wheat) and non-annotated sRNA dependent on the analysis and annotation of the five sRNA libraries; the spatiotemporal expression patterns ofthese identified miRNAs in different organs and developmen stages were analyzed and thetarget genes of these miRNAs were predicted.79miRNA was identified in this study, whichcontains24know miRNA and55novel miRNA. These55novel miRNAs in this studypositioned to54miRNA loci of wheat genome and have been included by the miRBase(version21, june2014; http://www. mirbase. org). The24known miRNA (belonged to15miRNA families) identified in this study located to18miRNA loci in wheat genome;9ofthem (9MIRNA loci) has been registered previously, and the remaining15miRNA (9MIRNA loci) were the first identified in wheat. Conservative analysis found that13(16MIRNA loci) of them belonged to the highly conserved miRNA families, while2(2MIRNAloci) of them belonged to the moderately conserved miRNA families. Based on the stricttarget gene predication standard in previous report, target genes of the identified knownmiRNA and novel miRNA in this study were predicted. The results show that a total of15known miRNAs and37novel miRNAs have predicted target genes which involve in widelybiological functions.3. The expression levels of randomly selected representative miRNA species in the fivetissues were further determined through quantitative real-time RT-PCR, firstly,9miRNAsincluding5known miRNAs,3novel miRNAs and1candidate miRNA were randomlyselected for abundance analysis. polyA qRT-PCR. The result of those two methods showed apearson coefficient (R2=0.892, P <0.01), which indicated that our sequencing result werereliable. After that, spatiotemporal expression profile of identified24known miRNAs and55novel miRNAs in the5organs or development stage were analyzed based on sequencing data.Most miRNAs including known ones and novel ones showed tissue-prefer expression tovarying degrees. Logarithm (log2) of the fold changes of known miRNAs in5samples wasvarying from-4.6to5.2, while novel miRNAs vary from-8.6to7.6. Four known miRNAfamilies (miR160, miR164, miR166and miR169) and22novel miRNAs prefer to express inseed. Twelve of the22novel miRNAs were specificly expressed in seed, their logarithm(log2) of the fold changes between1.0and7.6. From5–d seeds to20–d seeds, miR164andmiR160increased in abundance, whereas miR169decreased. This result suggests that thesemiRNAs might have coordinating functions in the different developmental stages of wheatseed. The potential targets of these known miRNAs were predicted. The expression patternsof these miRNAs were closely related to their functions, which also suggested that novelmiRNAs might participate in regulating wheat seed development and metabolism.Eightknown miRNA families (miR156, miR172, miR168, miR396, miR159, miR398, miR1318and miR167) and28novel miRNAs prefer to express in flag leaves. Logarithm (log2) of the was between0.1~5.2.Putative targets of these miRNAs encode a wide range of proteins,including protein kinase, glycosyltransferase, functional protein (Histone H2B.1), and otherenzymes, which are involved in diverse developmental and metabolic processes. Targetprediction result that flag leaves expression preferred miRNAs showed that miRNA involvedregulation network in flag leaves is more complicated than in seed.4. In order to identify tasiRNA, partial sequence of TAS3a and TAS3b, includingcomplete tasiRNA position region, were cloned from wheat (Triticum aestivum L.) cultivarXiaoyan6. Their lengths were698bp and650bp, respectively. Blast result indicated thattasiRNA position region was conserved. We blasted cloned TAS3a and TAS3b gene with oursRNA datebases and found12and13phased sRNAs matching their sequence in both strands.Two tasiARFregulated mRNAs encoding auxin response factors (ARF) were generated fromD6(+) and D7(+) positions in TAS3a，and one tasiARF were generated from D4(+) positionsin TAS3b. The most abundant tasiRNAs were generated from D6(+) and D4(+) positions inTAS3a and TAS3b, followed by D4(-) and D7(-) positions in TAS3a and TAS3b,respectively.The tasiRNAs derived from D4(-) position in TAS3a and the D7(-) position inTAS3b targeted to resistance proteinr and Cold-responsive protein, This results indicatedTAS3gene could take part in plant growth and development and may be involved instress-tolerance.5. To study the feasibility of BSMV for miRNA functional analysis, artificial miRNA ofwheat PDS gene (AmiPDS) was connected to BSMV. Photobleaching phenotype wasobserved on wheat leaves after wheat infected by BSMV-amiRPDS at seedling stages.Semiquantitative PCR was showed that mRNAs of PDS gene were much lower inphotobleaching leaves compared with control. Both forward and reverse sequences oftae-miR156precursor were connected to BSMV individually. The BSMV-miR156-F andBSMV-miR156-R were transfected in wheat at seedling stages,which displayed two differentphenotypes relative to growth period, one featured with no heading and vegetative growthonly, the other characterized with delayed heading for7-10days. Abundance of tae-miR156in leaves of transfected wheat exhibiting above phenotypes was much higher than control byreal-time PCR detection. In addition, further statistic analysis show that, tae-miR156abundances in plants infected by BSMV-miR156-F were significantly higher than those inplants infected by BSMV-miR156-R, suggesting that BSMV-miR156-F was more effective infuntional study of miR156.The research increased wheat miRNA information in miRBase, and revealed thespatiotemporal expression profile of the identified wheat miRNA. This provide fundamentalbasis for further study on miRNA functiona in wheat growth, development, especially regulation functions and network of flag leaves and seeds growth and development.