Identification And Analysis Of MicroRNAs At 4 Different Floral Developmental Stages Of Phyllostachys Edulis

Moso bamboo(Phyllostachys edulis),is the most widely distributed and planted bamboo specie which is an important economic crop with great economic value and huge production.The characteristics of moso bamboo flowering are scarcity,randomness and uncertainty.The flowering time of moso bamboo was different even if they were in the same area.The rhizome system and ground plants will become dead when moso bamboo flowers,leading to severe economic and ecological problems.Small RNA Illumina sequencing,bioinformatics analysis,q RT-PCR,gene cloning and transgenosis were used to select the key microRNAs(miRNAs)related to moso bamboo flower development and study the floral development mechanism.The major results are as follows:1.Illumina sequencing was used in libraries of small RNAs from no-flowering moso bamboo samples and flowering moso bamboo samples at different stages in order to identify known and novel miRNAs involved in the development of moso bamboo flowers.After filtering out the reads without small RNA sequences that ranged from 18 to 30 nt in length,a total of 18 761 783,23 055 864,15 304 493,17 497 326 and 16 874 894 reads were respectively got from the five libraries while small RNA species were respectively 7 684 089,11 325 554,6 470 328,6 701 286 and 6 678 093.409 known miRNAs,492 differentially expressed novel miRNAs and 29 miRNA*s were identified in moso bamboo.308 targets of 64 differentially expressed known miRNAs between no flowering samples and flowering samples were predicted.2.The differential expression and specifical expression analysis of mature miRNAs were performed between no flowering leaves and different flower developmental samples.We found that the expression abundances of many miRNAs in leaves were significantly higher than those at flower development stages.The number and species of exclusively expressed known miRNAs were also obviously greater than those at flower development stages.Functional annotation and GO enrichment analysis showed that many target genes of differential expressed miRNAs associated with the stimulus response and transcription factors.The expression patterns of miRNAs and target genes were examined by q RT-PCR.The results indicated that the expression profiles of miRNAs were negatively correlated with those of their targets.3.miR164 a,miR166 a,miR319 a and miR4414 b were selected to clone precursor genes.Full sequences of the 4 kinds of miRNA precursor genes were respectively obtained in moso bamboo for the first time.Besides,we have successfully constructed 4 sorts of overexpression vectors: p CAMBIA2300-miR164 a,p CAMBIA2300-miR166 a,p CAMBIA2300-miR319 a and p CAMBIA2300-miR4414 b.Agrobacterium-mediated transformation was used to transform miR164 a and miR166 a into Arabidopsis thaliana.So far we have got the T2 generation of transgenic plants,and obvious blossom delay and leaves shriveled phenomenen have been observed in the T1 generation of transgenic plants.According to the results of small RNA sequencing and differential expression profiles analysis for the moso bamboo,a great number of miRNAs information was acquired,and we found many key target genes of differentially expressed and specifically expressed miRNAs.Our research provided a scientific basis for the study on the molecular regulatory mechanism of flower development in moso bamboo.