Analysis On Small RNA,Identification And Expression Of MicroRNAs With Their Target Genes During Somatic Embrvogenesis In Japanese Larch (Larix Leptolepis)

Somatic embryogenesis is the major mean that large-scale propagation and popularizationof improved variety for conifers, and acts as a bridge for genetic engineering. It also has greatscientific meanings and application value in the study of cell totipotency, germplasmpreserving, renewing of forest tree and other fileds. However, many problems such as theinduction of embryogenic cell with difficulty, abnormal embryo with high ratio, andunder-synchronization during somatic embryogenesis are the bottleneck of the application.Until now, studies on conifers' somatic embryogenesis are commonly at physiology andbiochemistry levels, and many related genes are isolated and identified, which is not enough tosolve the problems that existed in the production and scientific research of somaticembyogenesis. Recent studies showed that, microRNA (miRNA) and other small RNAs(sRNAs) function at the upper level of gene regulation, play important regulatory roles duringembryogenesis. In this study, embryogenic cell and somatic embryo at eight developmentalstages from cell line O638＃of Larix leptolepis were collected, which were used to theconstruction of small RNA library and sequenced by Solexa tecnique. Bioinformatic methodswere applied to the analysis of sRNAs, identification of miRNA, prediction of miRNA target,identification of siRNA, degradation and modification of miRNA. miRNA targets werevalidated and miRNA precursors were cloned by RACE. Using qRT-PCR, expression analysesof miRNAs, miRNA precursors and miRNA targets were conducted during eightdevelopmental stages of somatic embryogenesis in larch. In addition, the expression levels ofRDR2and RDR6were monitored during eight developmental stages of larch. The main resultsare as follows:1. Sequencing results showed that overall20427626redundant sequences from2485006non-redundant sequences were obtainded during larch SE,, and18555811including2201297non-redundant sequences ranging from16-~30-nt were used for further analysisafter clearing non-sense sequences. The ratio of sequences matching to the known RNAs was 44.44%and9.61%to redundant sequences and non-redundant ones, respectively, of which,the number of sequence that matched to the repeat was the highest, followed by rRNA.Theseunknown sequences, exhibited a length bias of24-nt, representing20.8%and56.9%toredundant sequences and non-redundant one, repectively, indicating that conifers are capableof producing24-nt sRNAs with high diversity.24-nt sRNAs exhibited the lowest redundancy,and the average copy of per non-redundant sequence was only1.82, indicating the highdiversity of24-nt producing loci. The category of sRNAs differed along with thedevelopmental stages, which might be related to the distinct expression levels of RDR2andRDR6.2. We identified83conserved miRNAs from35families, of which12families (e.g.miR946, miR947, miR950, and et al.) were might specific to conifers. In total of16novelmiRNAs, and14plausible miRNA candidates were discovered, which was specific to larchwith a high proportion (96.3%). Of these,27precursor sequences belonging to conservedmiRNA and27sequences to novel miRNA or candidates were obtained. The non-maturesequences of the precursor were different among distinct species, and the similarity related tothe relationship of species. We predicted110target genes using bioinformatics, and thesegenes participated in many respects of growth and development in plants. We validated elevenof them by5' RACE, and the cleavage sites usually were between the10thand11th.3. The minor expression peak of many miRNAs was at late single embryo or earlycotyledonary embryo, the lowest level was at middle cotyledonary embryo, while the majorpeak was at late cotyledonary embryo, which consistent with the physiology events ofcotyledon formation, embryo postripeness, and embryo dormancy. However, the expressionmodel of miRNA precursor was differed from that of miRNA, and precursor expression withmuch smaller extent than that of miRNA. The expression of mature miRNAs did not becorrelated to precursor miRNAs linearly, which might be regulated at multiple levels. Themajor expression peak of miRNA targets was at late single embryo or early cotyledonaryembryo, while the lowest level was at late cotyledonary embryo. The abundance of miRNAtagets conversely correlated with miRNA content, but only the correlation of miR390-TAS3 was significantly. In addition,"co-expression" phenomennon was observed during somedevelopmental stages, indicating the complex interaction between miRNA and its target.4. Trans-acting siRNAs (ta-siRNAs) generated from TAS3triggered by miR390wereidentified. The abundance of ta-siRNAs varied distinctly, ranging from0~320, of which threeta-siRNAs (3'D4,3'D10, and3'D16) showed high copies. Of these,3'D10was predicted totarget the ARF. These three ta-siRNAs reached its' peak level in the mature embryo, while theabundance decreased dramatically after the embryo germination, and kept low level during thelater growth and development. In addition, two novel ta-siRNA loci were discovered andmany ta-siRNAs were identified. Several MIR loci had dual functions, which could produceboth miRNAs (21-~22-nt) and long sRNA (23-nt~26-nt). The ratio of long sRNA to totalsRNAs conversely correlated with the conservation of MIR genes.5. The identification of many truncated miRNA sequences, suggesting that miRNAscould be degraded in either3' to5' or5' to3' direction, and the degradation frequency from3' to5' was higher than that of5' to3' for most miRNAs, indicating the miRNA degradationmight be regulated. The addition of adenine, uridine, and cytidine to the3' end of miRNAswas globally presented, and the subtle variability in isomiR expression might be regulated andbiologically meaningful. miR397a and llemiR-7were enriched for3' cytidine additions duringSE development (as was miR950b-5p in seedling), implying that the addition of cytidines tospecific miRNAs is biologically meaningful in plant development. The addition of adenine,uridine, and their combinations might be associated with3' to5' miRNA degradation.These results showed conifers have the conserved pathway of small RNA. Not onlyconserved miRNAs that common to land plants are expressed, but also conifer-orgymnosperm-specific miRNAs are existed, and larch-specific miRNAs are evolved in larch.These miRNAs and their targets expressed distinctively during somatic embryogenesis. In all,miRNA, ta-siRNA and long sRNA might participate in the complex regulation during somaticembryogenesis of larch.