Excavation And Functional Analysis Of The MiRNAs Related To Herbaceous Peony Flower Color Formation

Herbaceous peony(Paeonia lactiflora Pall.)is one of the well-known traditional flowers which has the reputation of "prime minister in flowers" since ancient times.Because of its high ornamental value,it is widely used for plant landscaping and garden greening and is widely cultivated in many countries around the world.Color is one of the most important traits for ornamental plants,which directly affects the ornamental value and commercial quality of plants.Herbaceous peony is rich in color,which can be divided into 9 colors.However,the current researchs on the color of herbaceous peony are mainly focused on the determination of pigments,the cloning and expression analysis of structural genes and the analysis of transcriptome sequencing.Although herbaceous peony color formation of metabolic and transcriptional regulation mechanisms are clear,the miRNA-mediated post-transcriptional regulatory mechanism is unknown.It is of great significance to cultivate the new varieties of rare and colorful cultivars,and to clarify the mechanism of the formation of miRNAs and the mechanism of herbaceous peony.To this end,the study used the red outer-petals,the yellow inner-petals herbaceous peony complex colors varieties 'Jinhui' as the material,used miRNA sequencing to identify the differentially expressed miRNAs in outer and inner petals,screened candidate miRNAs and their target genes for the color formation of petals and cleared the key miRNAs regulated the post-transcriptional mechanism of color formation.The main results were as follows:(1)It was found on the impact of the color formation of physical and chemical factors that the distribution of pigment,the shape of skin,the pH value of cell fluid,the content of organic acid and the content of mineral element did not have significant correlation with the formation of inner and outer petals color.Then,the inner and outer petals miRNA libraries were constructed and sequenced.After removing the low-quality reads,more than 96.54%of the high-quality clean reads were obtained from the 6 samples of the inner and outer petals.After splicing,a total of 1,756,525 and 2,045,228 miRNAs,a total of 331 known miRNAs and 66 novel miRNAs were expressed in 6 groups of samples.A total of 163 known differentially expressed miRNAs and 28 novel differential expression miRNAs were obtained by differential analysis.Then,in the known differentially expressed miRNAs,80 miRNAs were down-regulated and 83 miRNAs were up-regulated,In the novel differentially expressed miRNAs,18 miRNAs were down-regulated and 10 miRNAs were up-regulated,and the function of the target gene was annotated as biochemical metabolic pathway and signal transduction pathway.The subcloning and expression patterns of miRNA precursor sequences were verified by randomization to verify the accuracy and reliability of miRNA sequencing results.(2)It was found that the improved multi-step extraction of herbaceous peony petals was better by comparing three kinds of small RNA extraction methods,and the extracted miRNAs were of high quality and good stability,which could meet the research of herbaceous peony miRNAs.By combining the miRNA sequencing results with the transcriptome data,miR156e-3p,miR828a,miR156d,miR2616 and Novel-miR25 were screened according to the function of the target gene.The candidate miRNAs were selected to control the color formation of herbaceous peony flowers.The expression pattern of miR156e-3p and its target gene Unigene 10411 was found to be negatively correlated with the expression pattern of candidate miRNA and its target gene in petals at different developmental stages by real-time quantitative PCR(qRT-PCR),The cloning alignment of miR156e-3p target gene Unigene10411 was similar to that of other species SPL(squamosa promoter-binding protein),which was named PISPL1.(3)The expression vector of miR156e-3p precursor gene was constructed.The vector was introduced into Agrobacterium bacteria by using liquid nitrogen freeze-thawing method.The transgenic plants were obtained by using Arabidopsis thaliana inflorescence impregnation method,and GUS staining,PCR amplification and qRT-PCR detection were used to screen and identify positive plants.The transgenic Arabidopsis thaliana lateral branches showed red in the bolting stage,which was mainly due to the accumulation of anthocyanins.In addition,qRT-PCR showed that miR156e-3p was negatively correlated with SPL gene expression in transgenic Arabidopsis thaliana strain,while SPL gene and DFR gene expression also showed a negative correlation trend,indicating that herbaceous peony miR156e-3p can regulate SPL affected the expression of DFR to regulate plants color formation.