Identification And Expression Analysis Of MiR171 And Target Gene SCL6 During Somatic Embryogenesis In Lilium

Somatic embryogenesis is vital to the bulb propagation, germplasm preservation as well as genetic traits improvement in Lilium. MiRNA is a key regulation factor in plant somatic embryogenesis process, only few miRNA related to somatic embryogenesis have been clarified. miR171 and SCL6 in Lilium pumilum and Lilium davidii var. unicolor were both cloned; RLM-5’RACE technique were used for identifying interaction sites between miR171 and SCL6; qRT-PCR technology was used to investigate the expression patterns of miR171 and SCL6 in different developmental stages of somatic embryos, and the expression level relation between miR171 and SCL6 were analyzed; preliminary prediction of miR171 endogenous target mimics were finished using bioinformatics method, in order to lay foundation for further research on the regulation mechanism of miR171 during somatic embryogenesis in Lilium. The main results are showed as follows:1. Clone of Ipu-miR171a/b and lda-miR171a/c:Stem loop RT-PCR were used to clone the mature sequence of lpu-miR171a, lpu-miR171b, lda-miR171a and lda-miR171c.2. Clone and sequence analysis of target gene SCL6:Target genes LpSCL6-Ⅰ, LpSCL6-Ⅱ and LdSCL6-Ⅲ were cloned and bioinformatics analysis was employed, the results showed that the number of the same amino acid sequence between target gene SCL6 and AtSCL6 is as high as 62.8%; LpSCL6-Ⅰ, LpSCL6-Ⅱ and LdSCL6-Ⅲ respectively have high homology with false bromegrass, date, and sesame; Proteins encoded by targets are hydrophilic and unstable; LpSCL6-Ⅰ, LpSCL6-Ⅱ and LdSCL6-Ⅲ respectively containing different amounts of alpha helix, extending chain, beta turn and randon coil.3. Identifying interaction sites of miR171 and SCL6RLM-5’ RACE technique was used to identify the target sites, results showed that lpu-miR171a, lda-miR171a and lda-miR171c only have one cleavage site, and the frequency of this cutting site is 20/20; lpu-miR171b has 2 cleavage site, cutting frequencies were 17/20 and 3/20.4. Expression analysis of miR171 and targets SCL6 during somatic embryogenesis in LiliumIn embryonic callus stage, lpu-miR171a has the highest expression level, in the torpedo-shape embryo stage lpu-miR171b, lda-miR171a and lda-miR171c respectively have a peak expression level. lpu-miR171a and lpu-miR171b exhibit negative regulatory relationships with targets; lda-miR171a and lda-miR171c showing positive regulatory relationships with targets.5. Prediction of endogenous target mimicsPrediction of miR171 eTMs was finished, results showed that lpu-miR171 have five eTMs, they are>c25984_g1_i1,>c30177_g2_i2,>c48244_g1_i1,>c49041_g1_i3, and>c61912_gl_i1; and lda-miR171 have two eTMs, they are>c61215_g1_i1 and>c77568_g1_i2.