The Function Analysis Of ETM-miR171-SCL6 Module Regulating Somatic Embryogenesis In Lilium Pumilum DC.Fisch.

Somatic embryogenesis in plants,which exhibits the advantages of universal occurrence,genetic stability,and suitability as an exogenous gene transfer receptor system.It is of great significance to the improvement of genetic traits and high-quality bulb breeding in Lilium industry.The previous research of our group was clear that miR171 plays an important role in the somatic embryogenesis of Lilium,but its mechanism of regulating somatic embryogenesis of Lilium and its internal regulatory network are not yet clear.This paper taking miR171 as the starting point for research,used overexpression,point mutation,STTM artificial silencing and CRISPR/Cas9 gene editing technology to clarify the functions of lpu-e TM171a/b,lpu-miR171a/b and the target gene Lp SCL6-II/I in Lilium pumilum DC.Fisch.The e TM-miRNA-Target regulatory network in the process of Lilium somatic embryogenesis was constructed,and revealed the regulatory relationship between e TM-miR171-SCL6 modules,which is of great significance for clarifying the mechanism of lily somatic embryogenesis.The main research results are as follows:1.A stable and efficient genetic transformation system with embryogenic callus of Lilium pumilum DC.Fisch.was established,and the genetic transformation efficiency reached 29.17%.The CRISPR/Cas9 gene editing system was successfully applied to Lilium,and Lp PDS gene in Lilium pumilum DC.Fisch.was knocked out.Sequence analysis in the transgenic lines revealed various mutation patterns,including base insertion,deletion and substitution.The successful application of stable and efficient genetic transformation system and gene editing technology in Lilium have laid an important foundation for gene function research and germplasm improvement in Lilium spp.2.In Lilium,lpu-miR171 can cleave the target gene Lp SCL6 and inhibit its expression.The obtained lpu-miR171a/b overexpression Lilium plants showed a phenotype that the development of the root system was blocked and the number of leaves was reduced.In contrast,silent plants have strong roots and more leaves.Silencing lpu-miR171a/b promoted the formation and development of Lilium somatic embryos,and significantly shortened the development of somatic embryos.The starch content of lpu-miR171a/b silent lines is significantly higher than that of wild-type,and the key cell cycle genes Lp CYCB1 and Lp CYCD3 were significantly up-regulated,and the effect of miR171 b was more obvious.3.To further clarify the function of lpu-miR171,the function of its target gene Lp SCL6 in lily somatic embryogenesis was studied.The results show that Lp SCL6-I and Lp SCL6-II proteins are both nuclear proteins.In Lilium,Lp SCL6-II/I can regulate lpu-miR171a/b with positive feedback.The Lp SCL6-II/I overexpression lines have advanced somatic embryogenesis,shorter development cycle,and higher cotyledon-shaped embryo formation rate.This was accompanied by significant up-regulation of key cell cycle genes Lp CYCB1 and Lp CYCD3.Therefore,overexpression of Lp SCL6-II/I can positively regulate Lilium somatic embryogenesis.At the same time,the starch synthase genes Lp AGP1,Lp SSS1 and Lp GBSS in the somatic embryos of the Lp SCL6-II/I overexpression lines were significantly up-regulated,while the key genes of starch metabolism enzymes Lp AMY and Lp BAM were significantly down-regulated.4.In order to analyze the regulatory network of lpu-miR171 in the process of Lilium somatic embryogenesis,we conducted study on the regulatory effect of e TM on lpu-miR171 during Lilium somatic embryogenesis.The full length c DNA of lpu-e TM171a/b was cloned from Lilium pumilum DC.Fisch.The length of lpu-e TM171 a is 1383 bp,which encodes 460 amino acids.Amino acid sequence alignment revealed that the homology with Phoenix dactylifera was high,reaching 79.29%,phylogenetic analysis found that it is closely related to Glycine max and Abrus precatorius.The length of lpu-e TM171 b is 1869 bp and encodes 622 amino acids.It has the closest relationship with Elaeis guineensis and the homology is high,reaching 68.24%.Lpu-e TM171a/b proteins are located in the nucleus and belong to nuclear proteins.The lpu-e TM171a/b and point mutation overexpression vectors were constructed and transformed into Lilium.In the late somatic embryogenesis of lpu-e TM171 b overexpression lines,a few somatic embryos were observed to germinate to form cotyledon-shaped embryos.High expression of lpu-e TM171a/b has a negative regulatory effect on the corresponding target lpu-miR171a/b,and the expression level of the corresponding target gene Lp SCL6-II/I is significantly increased.However,the expression of miR171a/b and target gene Lp SCL6-II/I in the point mutation overexpression lines OE-Me TM171a/b did not change significantly compared with the control.The above results confirm that lpu-e TM171 can be used as a bait molecule for miR171,which binds lpu-miR171 to prevent the cleavage of its true target gene Lp SCL6.In summary,silencing lpu-miR171a/b or overexpressing the target gene Lp SCL6-II/I showed early somatic embryogenesis,shorter somatic embryo development cycle,and higher cotyledon-shaped embryo formation rate,accompanied by increased starch content.High expression of lpu-e TM171a/b can affect the activity of lpu-miR171a/b and thus affect the expression of Lp SCL6-II/I.This study clarified the mode of action of e TM-miR171-SCL6 module involved in regulating somatic embryogenesis of Lilium pumilum DC.Fisch.