Identification And Differentially Expressed Analysis Of MicroRNAs At Different Dormancy Stages Of Pear Flower Buds

Pear flower as Chinese traditional flowers which has a long history of cultivation in China.Pear flower not only has a very high ornamental value,but also has a deep cultural background in Chinese traditional culture.Flower bud dormancy is indispensable to survival over winter and to regrowth and development in the following year.Flower buds from the dormancy to the normal growth stage need to accumulate a certain low temperature,and sometimes the lack of chilling requirement results in flower buds cannot complete the normal dormancy establishment and release process.MicroRNAs（miRNAs）are a class of small,endogenous,non-coding RNAs that take part in regulating genes through inhibiting target gene translation at the post-transcriptional level in plants.More evidences have shown that miRNAs play critical roles in plant cell division,morphogenesis,hormone secretion,signal transduction as well as responses to stresses.However,the role of miRNAs in pear flower bud is not clear.Therefore,it is of great significance to study the molecular mechanisms involved in the transition of flower bud dormancy.In this study,the flower buds of‘Huanghua’pear were used as material to identify five dormant miRNAs at different times through highthroughput sequencing of the HiSeq 2500 platform and bioinformatics technology,and miRNAs related to dormancy were identified.Quantitative real-time PCR（qRT-PCR）was used to validate the expression levels of miRNAs.Selected appropriate reference genes for normalization of miRNAs expression in pear flower bud during dormant phase.Finally,the mature novel miRNAs were cloned and structure were analyzed.The main results as follows:1.Small RNA libraries were constructed using flower buds from five stages of dormancy,followed by high-throughput sequencing using Solexa technology.Five samples obtained 28,5 78,465、23,617,286、30,721,346、31,569,393、28,127,091 clean reads,of which small RNAs with a length of 24nt accounted for more than 50%,indicating that dormant flower buds were mainly 24nt in length.These sequences were aligned with the pear genome and miRbase database for BLAST sequencing.A total of 152 known miRNAs and 209 novel miRNAs were identified,belonging to 38 and 45 miRNA families,respectively.There were significant differences in the expression levels of 56 known miRNAs in 16 families,indicating that these miRNAs may be associated with flower bud dormancy in pears.2.Using bioinformatics prediction methods,Target Finder software was used for target gene prediction.332 target genes corresponding to 116 known miRNAs and 154 target genes corresponding to 42 new miRNAs were obtained.The results showed that the target genes involved in transcriptional regulation,hormone signaling,growth,development processes and so on.It shows that bud dormancy of pear is a complex physiological process and needs the coordination of genes with multiple functions.3.The expression level of 14 differentially expressed miRNAs and target genes in five samples were analyzed by qRT-PCR technique.The results showed that the qRT-PCR results of 13 miRNAs were in accordance with high-throughput sequencing results,which proved that the sequencing result were reliable.Generally speaking,the expression level of 10 miRNAs in the eco-dormancy were significantly higher than that in the endodormancy period,and the overall expression level of the miRNAs increased.Among them,the expression level of miR159 decreased from deep dormancy to endo-dormancy release stage,then reached the lowest value when the endo-dormancy was released.After the release of endodormancy,the expression level increased during the period of ecodormancy.qRT-PCR results showed that the expression of target gene GAMYB was negatively correlated with miR159 in the target gene of miR159.Therefore,miR159 may play a regulatory role in the GA pathway of endo-dormancy release in pear flower bud by inhibiting the expression of the target gene GAMYB.The target genes of miR156 and miR172 were SPL and AP2 transcription factors,both of which may be involved in the flowering process of pear after eco-dormancy release.The expression level of miR160 increased from the deep dormancy phase to the endodormancy release phase and then decreased.The expression level of miR160 was significantly upregulated during the period of eco-dormancy release.This change of miR160 may be related with the transformation of the whole dormancy state of the flower bud.4.In five different periods of dormancy buds as material,with three commonly used houskeeping genes（U6 snRNA,5.8S rRNA,5S rRNA）and 12 miRNAs from the sequencing as reference candidate genes,The expressions of 15 candidate reference genes by qRT-PCR method in pear flower bud samples were investigated and their stabilities were analyzed by geNorm,NormFinder and BestKeeper.The results showed that miR3.0₂2782 was the most stable single reference gene in different dormancy periods of pear flower bud,the most stable combination of reference gene was miR3.0₂2782 and miR171.This study provided a basis for accurate detection the expression of miRNAs during dormant phase of pear flower bud.This indicated that in different stages of dormant pear flower buds,the use of miRNA as internal reference gene for qRT-PCR was more stable than some commonly used rRNA and snRNA.5.High-throughput sequencing was used to predict and screen out novel miRNAs,the novel miRNAs with the top fifteen expression levels and differential expression were subjected to mature clone,precursor secondary structure analysis,target gene prediction and qRT-PCR assay.The target genes were predicted and qRT-PCR test were carried out for the top ten expressed and differentially expressed genes.The results showed that 13 miRNAs had PCR amplification bands,and mature sequences were consistent with high throughput sequencing results,demonstrating the accuracy of high-throughput sequencing.The result of qRT-PCR showed that two novel miRNAs（miR1226.0₅446 and miR156.0₁0377）were significantly different in eco-dormancy stage,presumably related to the release of eco-dormancy.