Spationtemporal Expression Of MiRNAs In Leaf And Shoot Apical Meristem Of Winter Wheat

Wheat can be divided into winter wheat and spring wheat.Winter wheat needs certain time cold exposure to switch from vegetative growth to reproductive growth,named as vernalization,while spring wheat does not need vernalization.The yield of winter wheat accounts for more than 90% of total wheat in our country,indicating its important position in China's national economy and people's livelihood.The physiological processes,the key regulatory genes and their interactive associated with wheat vernalization are relatively studied well.MicroRNA is a kind of small RNA which negatively regulates target genes by post-transcriptional regulation.Most target genes of conserved miRNA are transcription factors,which regulates the expression of multiple downstream genes.Therefore,the involvement of miRNAs in regulatory network is particularly important for plant development.The roles of miRNAs in Arabidopsis and rice growth and development are clear,while studies about wheat miRNA were mainly focused on stress response and wheat grain development.The regulation of miRNAs in winter wheat growth and development is not clear.In the official miRNA database miRBase?Release 22.1?,rice has 738 mature miRNAs;Arabidopsis has 428 miRNAs;Brassica has 525 miRNAs,wheat only has 125 miRNAs.It is clear that identification of wheat miRNAs remains to be further studied.In order to identify other potential wheat miRNAs and explore the regulation mechanism of miRNAs in the growth and development of winter wheat,we used winter wheat "Shimai 22" as experimental material and studied the small RNA profiles of wheat leaf and shoot apical meristem at three-leaf stage,wintering period,regreening stage and flower primordium differentiation stage.Totally,8 small RNA libraries were generated and analyzed,differentially expressed miRNAs were identified and 10 miRNA target genes were validated.The main findings are as follows:?1?By comparison with all plant miRNAs deposited in miRBase database,373 known miRNAs belonging to 289 miRNA families were identified in this study.The 23 most conserved miRNA families in angiosperms?miR156/157,miR159,miR160,miR161,miR162,miR164,miR165/166,miR167,miR168,miR169,miR170/171,miR172,miR319,miR390,miR391,miR393,miR394,miR395,miR395,miR397,miR398,miR399,miR408?and the monocotyledonous conserved miR444 were all detected.?2?According to the miRNA anotation criteria,82 novel miRNAs were identified in this study.Based on sequence homology,38 of them were found already reported in other related literature,but the sequences have not been deposited in miRBase.Therefore,44 novel miRNAs were found for the first time in this study.?3?Forty five differentially expressed known miRNAs and 31 novel miRNAs were found in leaves.Among them,miR9662a-3p and novel miR2079 were abundantly expressed in the three-leaf stage;miR156a-5p,miR165a-3p,miR5200-5p,miR9778 and novel miR1419 had high abundance in the wintering period;miR172a-5p,miR172e-3p,miR167 a,miR408d,miR528-5p,miR9672 b,novel miR1154 and novel miR1875 were highly induced in flower primordium differentiation stage.Fifty differentially expressed known miRNAs and 35 novel miRNAs were found in shoot apical meristems.Among them,miR9662a-3p,novel miR30 and novel miR1372 have high abundance in the three-leaf stage;miR156a-5p,miR167 a,miR5200-5p,miR9675-3p and miR9778 have high abundance during the wintering period;miR172a-5p,miR172e-3p,miR394 a and miR9672 b were highly induced during flower primordium differentiation stage.These results indicate that miRNAs play essential roles in different tissues at different growth and development stages.Our results are consistant with previous finding that miR156 and miRNA5200 inhibiting wheat flowering,miR172 and miR408 promoting wheat flowering process.?4?Target genes of all miRNAs in wheat were predicted,and target genes of 224 known miRNAs and 48 novel miRNAs were predicted.Fifteen predicted target genes were selected to be validated using RLM-5?RACE method,and 10 target genes were verified,including two highly conserved miRNA target genes.TRIAE_CS42₆AL₄72660_AA1524550.1?scarecrow-like protein?is a target gene of miR171a-3p,TRIAE_CS42₂AL₀97448_AA0324210.1?AP2-like ethylene-responsive factor?is a target gene of miRNA172e-3p.Four target genes of 4 wheat species specific miRNAs were also verified.TRIAE_CS42₅BS₄23630_AA1380910.2?putative disease resistance protein?is the target gene of miR1127 a,TRIAE_CS42₄DL₃43545_AA1136340.2?vulgare mRNA for predicted protein?is the target gene of miR5181,TRIAE_CS42₆DS₅42718_AA1728780.1?Transcription termination factor?is the target gene of miR9662a-3p,TRIAE_CS42₆BS₅13439_AA1641660.1?protein release factors?is the target gene of miR9674;in addition,4 target genes of 3 novel miRNAs were also verified,TRIAE_CS42₂AS₁13055_AA0350560.2?tauschii sialyltransferase-like protein?is the target gene of novel miR84,TRIAE_CS42₃B₂20641_AA0713280.1?integrator complex subunit 9 homolog?is the target gene of novel miR692,TRIAE_CS42₆DS₅43340_AA1738640.3?vulgare mRNA for predicted protein?and TRIAE_CS42₆BS₅14840_AA1664920.1?transcription termination factor?are target genes of novel miR1356.For the first time,8 target genes of four wheat-specific miRNAs and three novel miRNAs were verified in this study,which laid foundation for further exporing the regulatory function of these miRNAs.A total of 455 wheat miRNAs were identified in this study,which enriched the knowledge of wheat miRNA.The temporal and spatial expression of these winter wheat miRNAs were studied,and 8 target genes of wheat species specific miRNAs were verified.These results indicate that miRNA is a kind of key non-coding regulation factor that is involved in many biological processes during wheat growth and development.Our study provides valuable information for deep understanding the regulatory mechanism of miRNAs in winter wheat growth and development.