Characterisation and Expression Analysis of LdSERK1, a Somatic Embryogenesis Gene in Lilium davidii var. unicolor

Abstract

The Lanzhou lily (Lilium davidii var. unicolor) is a variant of the Sichuan lily of the lily family and is a unique Chinese 'medicinal and food' sweet lily. Somatic cell embryogenesis of Lilium has played an important role in providing technical support for germplasm conservation, bulb propagation and improvement of genetic traits. Somatic embryogenesis receptor-like kinases (SERKs) are widely distributed in plants and have been shown to play multiple roles in plant life, including growth and development, somatic embryogenesis and hormone induction. Integrating the results of KEGG enrichment, GO annotation and gene expression analysis, a lily LdSERK1 gene was cloned. The full-length open reading frame of LdSERK1 was 1875 bp, encoding 624 amino acids. The results of the phylogenetic tree analysis showed that LdSERK1 was highly similar to rice, maize and other plant SERKs. The results of the subcellular localisation in the onion epidermis suggested that the LdSERK1 protein was localised at the cell membrane. Secondly, we established the virus-induced gene-silencing (VIGS) system in lily scales, and the results of LdSERK1 silencing by Tobacco rattle virus (TRV) showed that, with the down-regulation of LdSERK1 expression, the occurrence of somatic embryogenesis and callus tissue induction in scales was significantly reduced. Finally, molecular assays from overexpression of the LdSERK1 gene in Arabidopsis showed that LdSERK1 expression was significantly enhanced in the three transgenic lines compared to the wild type, and that the probability of inducing callus tissue in seed was significantly higher than that of the wild type at a concentration of 2 mg/L 2,4-D, which was manifested by an increase in the granularity of the callus tissue.

Keywords: LdSERK1; Lilium davidii var. unicolor; gene isolation; phenotyping; somatic embryogenesis receptor-like kinase (SERK) gene.

Figure 1

Morphological observations of Lanzhou lily somatic embryo at different stages of development. (A) Globular stage under the autopsy microscope. (B) Heart-shaped stage under the autopsy microscope. (C) Torpedo-shaped stage under the autopsy microscope. (D) Cotyledon stage under the autopsy microscope. (E) Globular stage under tissue staining. (F) Heart-shaped stage under tissue staining. (G) Torpedo-shaped stage under tissue staining. (H) Cotyledon stage under tissue staining. Scale = 1 mm.

Figure 2

Transcriptional profiling of genes across four stages of development in somatic embryos. (A) Hierarchical clustering of all differentially expressed genes (DEGs) based on z−score normalised FPKM values. Blue indicates lower expression, and red indicates higher expression. (B) Differential expression of LdSERK1, LdSERK2 and LdSERK3 at four stages of somatic embryo development. (C) Distribution of differentially expressed genes in the KEGG pathway. (D) Gene function annotated Venn diagrams for NR, NT, KOG, KEGG and GO databases. (E) Genes differentially expressed between two random stages.

Figure 3

Analyses of the phylogenetic relationship, amino acid structure and LdSERK1 gene clone from lily. (A) Agarose gel showing the RT-PCR amplification of LdSERK1 coding DNA sequence (CDS) from lily. (B) Phylogenetic tree of LdSERK1 family proteins from selected plant species. The phylogenetic tree was constructed in MEGA6.0 software using the neighbour-joining method. (C) Prediction of the transmembrane structure of the LdSERK1 protein. (D) Motif analysis of the LdSERK1 protein using the NCBI domain. * It stands for the name of the genus of the species.

Figure 4

Subcellular localisation of LdSERK1 protein in onion. (A) Subcellular localisation of the GFP protein in onion epidermal cells, scale bar = 50 μm. (B) Subcellular localisation of the LdSERK1 protein in onion epidermal cells, scale bar = 50 μm.

Figure 5

Establishment of a scale VIGS system for the LdSERK1 gene and analysis of the assay. (A) Callus status after 45 days of mixed infection with TRV1 and TRV2. (B) Callus status after 45 days of mixed infection with TRV1 and TRV2-LdSERK1. Red arrows indicate a large area of healing tissue growth, yellow arrows indicate no healing tissue growth and white arrows indicate a small area of healing tissue growth. (C) CP and MP on TRV1 and TRV2/TRV2-LdSERK1 backbones in nascent callus tissues of both treatment groups were detected as 250 bp transcript segments. CP: coat protein, MP: movement protein. 1–3 refer to TRV1/TRV2, 4–6 refer to TRV1/TRV2-LdSERK1, 7–8 refer to ddH₂O and negative control. (D) RT-QPCR analysis of LdSERK1 gene expression in control (CK), negative (TK) and positive (TS). Lowercase letters indicate result of ANOVA (p > 0.05).

Figure 6

Molecular phenotype of the overexpression of the LdSERK1 gene in Arabidopsis thaliana and phenotypic analysis of the induction of cotyledons into callus tissues. (A) Phenotypic comparison of the LdSERK1 overexpression transgene and the wild type. (B) Differential expression of LdSERK1 in overexpression and wild-type lines by fluorescence quantitative PCR. (C) PCR detection of Hyg in overexpression and wild-type lines. (D) Callus tissue induced from seeds in overexpression lines using 2 mg/L 2,4-D. (E) Callus tissue induced from seeds in the wild-type lines using 2 mg/L 2,4-D. **** It represents significant differences (p < 0.01).