| Poplar is widely planted in the world as an important economic tree species for fast growth and easy propagation.Furthermore,the whole-genome of Populus trichocarpa had been sequenced,which made it as the model plant in research of woody plants.The shoot apical meristem(SAM),which is a crucial tissue located at the top of woody plants,can continually undergo differentiation.The SAM gives rise to all above-ground organs,such as the leaves,through cell division and differentiation.SAM development was controlled by a series of complicated molecular regulation networks,accordingly with phased characteristics of the SAM.In previous studies,the research of SAM in the woody plants was focused on the regulatory mechanism of plant hormone and transcriptional level.However,few reports about the regulatory mechanism of miRNAs,a vital post-transcription regulatory factor,are reported in the SAM of the woody plants.In this study,we used high-throughput sequencing to build two libraries derived from shoot tips and mature leaves tissue in Populus tomentosa.Besides the post-transcription regulatory mechanism of SAM,seed germination is also an important foundation for growth development and sexual propagation in the trees.Therefore,we also investigated the effects of different external environmental factors on seed germination in P.tomentosa.The main results are as follows.(1)A total of 23,808,908 raw reads were obtained,of which 11,566,051 and 12,242,857 sRNA raw reads were produced in SAM and leaf sRNA libraries,respectively.The overall size distribution of filtered sRNAs from SAM and leaf library was distributed from 18 nt to 30 nt,with the most abundant 21 nt sRNAs,which was consistent with the length distribution of sRNA in other angiosperms.(2)After using a BLASTN search for sequence similarity in the miRBase database,231 and 256 miRNAs were expressed in the SAM and leaf libraries,respectively.In addition,185 and 181 novel miRNAs were identified in the SAM and leaf libraries,respectively.(3)To identify putative targets for known and novel miRNAs,the web-based programs psRobot and TargetFinder were employed to hunt for potential miRNAs target genes.The results showed that a total of 1,796 and 1,507 genes were predicted to be targets of known and novel miRNAs,respectively.Among them,these target genes were related to binding,biological process and catalytic activity.(4)We analyzed the differences expression level between SAM and leaves,found that in comparison to leaves,in conserved miRNAs,14 miRNAs were significantly up-regulated in the SAM library,whereas the other 54 miRNAs were preferentially down-expressed in the SAM library;in non-conserved miRNAs,27 miRNAs significantly up-regulated in the SAM library,whereas,the other 30 miRNAs were preferentially down-expressed in the SAM library.Furthermore,we also analyzed the functions of target genes of differentially expressed miRNAs.The results showed that,in the known miRNAs,up-regulated miRNAs in the SAM library were associated with phytochrome interactions,leaf dyed and morphogenesis,whereas down-expressed miRNAs in the SAM were related with cell division,auxin and cytokinin response.In addition,in the novel miRNAs,up-regulated miRNAs in the SAM library were associated with photosynthesis,whereas down-expressed miRNAs in the SAM were related with RNA polymerase.The results demonstrated that SAM could regulate plant growth and development through suppressing the expression of the target genes of phytohormone-related miRNAs during the development process.(5)Ptc-miR166n,ptc-miR166o and ptc-miR166q,which were included in the family of ptc-miR166,were significantly down-regulated in the SAM library,and targeted several members of cell division related transcription factor family,class ?HOMEODOMAIN-LEUCINE ZIPPER(HD-ZIP).The results of q RT-PCR showed that an inverse relationship was observed between the expression of ptc-miR166 and its targets.It suggested that HD-ZIP could be the target genes of ptc-miR166,and ptc-miR166 could regulate the development of SAM by inhibiting HD-ZIP transcription factor.(6)Ptc-miR393a,ptc-miR393b and ptc-miR393c,which are included in the family of ptc-miR393,were significantly down-regulated in the SAM library,and target several members of auxin response related transcription factor family,transport inhibitor response 1(TRI1).The results of q RT-PCR demonstrated that there was an opposite expression pattern between the expression of ptc-miR393 and its targets.It suggested that TRI1 could be the target genes of ptc-miR393,and ptc-miR393 could play an important role in the response of auxin through suppressing the expression of the TRI1 transcription factor.(7)We investigated the effects of environmental factors on seed germination of P.tomentosa,such as temperature,salinity,pH and light intensity.The seed germinates rate reached the top when the day and night temperature was 23? and 18?,respectively.Meanwhile,seed germinate was significantly influenced by salt stress.The rate of seed germination keeps decreasing while the environmental salinity increasing.In addition,the pH also affect seed germination.The seeds germinated easier at neutral pH compared with those of acidic or alkaline environment.Moreover,within a certain light intensity,the seed germination rate increases with the higher light intensity.In summary,to explore the regulatory networks of miRNAs in the SAM and leaves of P.tomentosa,two sRNA libraries was constructed from SAM and mature leaf tissue.And,differentially expressed miRNAs and its targets were annotated by bioinformatics analysis.Furthermore,we used q RT-PCR to detect the expression patterns of miRNAs and its targets during development growth.In the meanwhile,to investigate the optimal environment for seed germination,we also treated seed with different environmental factors.Our results indicate that many known and novel miRNAs were associated with cell division,hormone response and development in the regulatory mechanism of the developmental SAM,and the optimal environment for seed germination.Our results provide valuable data for studying shoot tip growth and seeding reproduction in P.tomentosa. |
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