Identification And Expression Analysis Of MiR166 And Target Gene During Somatic Embryogenesis In Lilium

Somatic embryos have the advantages of large quantity,high reproduction rate,easy germination and seedling formation.However,there are still some problems such as the ability of gene storage to be preserved and the slow reproduction rate.The study on the mechanism of Lily somatic embryogenesis provides theoretical basis and practical significance for the rapid propagation,transgenic breeding,mutant screening and artificial seed development.The process of protein synthesis is actually the process of gene expression.The miRNA can negatively regulate the expression of plant gene by guiding the mRNA degradation of target gene and inhibiting translation.Therefore,the study of the expression pattern of mRNA and target gene in the process of somatic embryogenesis of lily helps to reveal the molecular regulation mechanism of somatic embryogenesis of lily,thus solving a series of problems in the process of somatic embryogenesis.MiR166 plays a important role in the development of plant roots,vascular bundles and leaf polarity.In this study,we research identification and expression analysis of miR166 and target gene during somatic embryogenesis in Lilium,and draw the following conclusions:1.Clone of lpu-miR166a/b/c,lda-miR166a/b/c and sequence analysis of target gene:Stem loop RT-PCR were used to clone the mature sequence of lpu-miR166a/b/c,lda-miR166a/b/c and bioinformatics analysis were used to perdict target genes.The target of lpu-miR166a/lda-miR166a is LMBR1.The target of lpu-miR166b/lpu-miR166c is LpIDDl and the target of lda-miR166b/lda-miR166c is LdHD-ZIP III.2.Identifying interaction sites of miR166 and its target genesRLM-5'RACE technique was used to identify the target sites,results showed that lpu-miR166a/lda-miR166a only have one cleavage site,and the frequency of this cutting site is 10/10;lpu-miR166b/lpu-miR166c only have one cleavage site,and the frequency of this cutting site is 8/10;lda-miR166b/lda-miR166c has one cleavage site,cutting frequencies were 10/10.3.Expression analysis of miR166 and targets during somatic embryogenesis in LiliumLpu-miR166a/lpu-miR166b/lpu-miR166c was negatively correlated with the expression pattern of its target gene,in which lpu-miR166a was almost expressed in the embryogenic preservation stage and peaked in the globular embryo.The expression level of lpu-miR166b/lpu-miR166c was very low in the stage of embryogenic preservation,and the expression level of lpu-miR166b/lpu-miR166c was very low in the embryogenic preservation stage,and the expression level of lpu-miR166b/With the maturation of somatic embryos,the expression of lpu-miR166c showed a gradual upward trend,and the expression of lpu-miR166b showed a tendency to decrease first and then increase,and the expression level of LpIDD1 was increased first and then decreased Lda-miR166a showed a positive correlation with the expression pattern of LdLMBRl,and the expression of lda-miR166a increased gradually with the maturation of somatic embryos,reached the peak at the time of cotyledon embryo and the expression of LdLMBRl was not significant.Lda-miR166b/lda-miR166c was the lowest in the period of embryogenic preservation.With the development of somatic embryos,the expression of lda-miR166c increased gradually,and the expression of lda-miR166b increased first and then decreased.LdHD-ZIP ? The expression level of ZIP ? decreased first and then decreased,indicating that lda-miR166b played an important role in regulating LdHD-ZIP ?.