Study On The Function Of MiR166A And Target Gene LMBR1 In Somatic Embryo Development As Well As Morphogenesis In Lilium

Lily is a bulbous flower with strong ornamental and high commercial value,which plays an important role in the international field.However,there are still many problems to be solved in bulbous propagation and germplasm innovation.Somatic embryogenesis has many advantages,such as high reproduction coefficient and low variation rate.It is an effective way to solve these problems,but the molecular mechanism of somatic embryogenesis is not clear.Our previous studies showed that miR166a was differentially expressed in the process of Lily somatic embryogenesis,but the function of its target gene LpLMBRl has not been reported in plants.In order to explore the function of lpu-miR166a and its target gene LpLMBR1 in the somatic embryogenesis and morphogenesis of lily,this study cloned the primary miR166a of Lilium pumilum DC.Fisch and its target genes.LpLMBR1 complete CDS region and bioinformatics analysis.With the help of the stable genetic transformation system of lily established by the research team,the role of miR166a in the somatic embryogenesis and morphogenesis of lily was initially clarified through techniques such as gene overexpression,knockout,and silencing.The function of LpLMBRl was first explored in plants.The main findings are as follows:1.The complete CDS sequence of LpLMBRl was obtained by cloning;The encoding protein of LpLMBRl is hydrophobic protein,which is located on the periphery of cell and is an inherent component of membrane,and plays a role of cell connection;LpLMBR1 is specifically expressed in leaves,roots and scales of Lilium pumilum DC.Fisch,among which the highest expression is in leaves and the lowest is in roots.The expression of LpLMBRl was significantly higher in the stage of embryo callus preservation and cotyledon embryo than that in the 15th day of induction,It is preliminarily determined that the gene plays an important role in the regulation of embryogenesis and the late stage of somatic embryogenesis.2.Over-expression and knock-out vectors of lpu-pre-miR166a and LpLMBR1,and silencing vectors of miR 166a were successfully constructed.Using the Agrobacterium-mediated genetic transformation system,both were knocked out and over-expressed in Lilium candida 'White Heaven',and at the same time,LpLMBR1 was knocked out in Lilium pumilum DC.Fisch.Through the identification of somatic embryo induction efficiency on transgenic plants,it was found that the scale of the LpLMBRl knockout plant was greener,the browning of the somatic embryo was severe at the initial stage,and the somatic embryo induction ability was significantly reduced.'cotyledon embryo' appeared at about 42 days after induction and formed earlier,The results showed that LpLMBRl was very important to maintain the normal morphology of Lily embryos.3.Histomorphological observations showed that after overexpression of miR166a,adventitious buds and adventitious root germination coefficients of the 'White Heaven' plant increased,and the root growth rate increased significantly.LpLMBR1 over-expressing lines showed leaf curl,weak adventitious bud germination ability,reduced number of lateral roots,and cotyledon fusion during adventitious bud regeneration.It can be seen that LpLMBR1 is not conducive to the morphological establishment of leaves and roots of lily and miR166a has a negative regulatory relationship with it.However,LpLMBR1 over-expressing lines had faster bulb growth rate than control plants,and the plants were short and stout.The regulation of bulb growth on LpLMBRl needs to be further explored.