Identification And Functional Analysis Of Small RNAs In Bamboo(phyllostachys Edulis) By High Throughput Sequencing

The Moso bamboo(Phyllostachys edulis) is one of the most important forestry resources and also plays essential ecological roles in southern China. Apart from that, bamboo is the fastest growing woody plant in the world with a unique rhizome-dependent system and exhibits monocarpic mass flowering. MicroRNAs(miRNAs) are approximately 21-24 nucleotides’ long small RNAs(sRNA), that function post-transcriptionally to regulate gene expressions by binding to complementary sites in target mRNAs and further leading to mRNA degradation or translational repression of the target gene. Plant miRNAs play critical roles in most of biological processes including development, differentiation, biotic and abiotic stress responses.In order to identify the function of miRNA in bamboo flowering and environment stimuli, the tissues of vegetative stage and reproductive stage of bamboo and striped bamboo were harvested. sRNA libraries were then constructed and sequenced by using the high-throughput Solexa technology. Bioinformatics and quantitative real-time PCR were performed to discover mi RNAs species and identify P. edulis sRNA population differential expression.Meanwhile, the small RNAs high-throughput parallel sequencing was used for identification of viruses in P. edulis, contigs were assembled from sequenced total siRNA and in one case spanned the entire genome. the existence of viruses in bamboo were identified by using RT-PCR and RACE. The main results in this study are as follows.More than twenty millions sequences were produced in each sRNAs libraries. All together, 249 known mi RNAs belonging to 45 miRNAs family were identified and 49 novel putative bamboo miRNAs with lower abundance were predicted by using bamboo EST sequences and full-length cDNA sequences available in public database. In bamboo, miR168 was detected as the most abundance species, conserved family miR156 and miR172 were also found to be highly expressed in this dataset at the same time. It was also interesting that non-conserved mi R535 and miR528 were abundant in bamboo.59 unigenes targeted by 22 known miRNAs families were predicted and no targets were predicted in bamboo for the remaining miRNA. The majority target mRNAs were predicted to encoding transcription factors. Additionally, only 22 out of 48 new miRNAs were identified their targets gene which encoded transmembrane protein, DNA methyltransferase, transcription factor ILR3-like and so on. It implied that the corresponding miRNA might participate in some specific development in bamboo.The majority reads(41.09%) of stripe bamboo small RNA libraries were classified tRNA with 31-35 nt in length, being the most represented class of non-redundant species. This result was not consistent with what was detected in other samples. it was supposed that these lsiRNAs were induced by pathogen infection or under specific growth conditions.By bioinformatics analysis in different samples, it was found that 31 known miRNA were involved in bamboo flowering, 31 known mi RNA were involved in bamboo stripes and 40 known miRNA were probably involved environment stimulate.Assembly of small RNA in all samples revealed that Rice tungro bacilliform virus and Citrus exocortis viroid, covered 91% and 86% of their respective genomes,were existed as endogenous virus in bamboo. 1992 bp RTBV fragments were successfully amplified and it was not existed polymorphism in different sample. Additionally, bamboo flowering was not immediately connected with virus infection.The expression levels of 14 frequently used housekeeping genes(reference genes) were assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples(root, stem, leaf, flower, and leaf sheath) and grown under various conditions. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. TIP41, followed by NTB and CAP2 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different samples. TIP41 is therefore the recommended reference gene for measuring gene expression in P. edulis.21 known miRNA and 3 novel miRNA were selected for expression pattern analysis by mi RNA LNA quanlity real time PCR, almost all of them showed differential expression in the different tissues analyzed. The expression of target genes SPL, AP2, HD-ZIPIII and PLA were negatively correlative with the expression of miR156, miR172, miR166 and miR528 in different tissues respectively.The miR168 and miRb17 showed differential expression in striped and normal bamboo by mi RNA LNA quantitative real time PCR analysis, which indicated they involved the stripe formation. Meanwhile,15 known miRNA and 3 novel miRNA showed differential expression in the vegetative stage and reproductive stage of bamboo.The miR156 and miR172 were expressed in inverse patterns in different tissues and different development stage. miR156 has the highest abundance in root, whereas miR172 has the least abundance. mi R156 declined from vegetative stage to reproductive stage, whereas mi R172 increased during this same period. miR156 and miR172 target two families of plant-specific transcription factors(respectively, SPL and AP2-like transcription factors) that cooperate to regulate bamboo flowering. The results presented here suggest the mechanism of bamboo flowering by which their expression is temporally coordinated like other plant previously reported.