Table 1

The sequences of primers used in this study"

Fig. 1

Cloning and full sequence of MsSQE1 from alfalfa A: PCR Amplification of MsSQE1; B: Full sequence of MsSQE1"

Fig. 2

Sequence alignment of the representative squalene epoxidase amino acid sequences from the indicated species"

Fig. 3

Phylogenetic tree of SQE protein sequences from alfalfa and other species"

Fig. 4

Analysis of the secondary structure of MsSQE1 protein A: The prediction of signal peptide of MsSQE1, C-score: Raw cleavage site score; S-score: Signal peptide score; Y-score: Combined cleavage site score; mean S: The average S-score of the possible signal peptide; D: Discrimination score; B: The prediction of transmembrane regions of MsSQE1; C: Analysis of the secondary structure of MsSQE1"

Fig. 5

Subcellular localization of 35S::MsSQE1-GFP in onion epidermal cell a-c: Images of onion epidermal cells expressing 35S::GFP taken under GFP fluorescence; d-f: Images of onion epidermal cells expressing 35S::MsSQS-GFP taken under GFP fluorescence; g-i: Images of dehydrated (0.3 g·mL-1 sucrose ) onion epidermal cells expressing 35S::MsSQS-GFP taken under GFP fluorescence. Bar=100 µm"

Fig. 6

Expression analysis of MsSQE1/MsSQE1ΔN25 by SDS-PAGE in E.coli M: Protein ladder; 1: Total protein expressed by E.coli. harboring pEASY-E2-Control(inducted 5 h with IPTG inducement); 2: Total protein expressed by E.coli. harboring pEASY-E2-MsSQE1ΔN25 (without IPTG inducement); 3: Soluble protein expressed by E.coli. harboring pEASY-E2- MsSQE1ΔN25 (inducted 5 h with IPTG inducement); 4: Inclusion membrane protein expressed by E.coli. harboring pEASY-E2- MsSQE1ΔN25 (inducted 5 h with IPTG inducement); 5: Total protein expressed by E.coli. harboring pEASY-E2- MsSQE1(without IPTG inducement); 6: Soluble protein expressed by E.coli. harboring pEASY-E2-MsSQE1(inducted 5 h with IPTG inducement); 7: Inclusion membrane protein expressed by E.coli. harboring pEASY-E2-MsSQE1 (inducted 5 h with IPTG inducement)); Arrows represent expression of MsSQE1 protein"

Fig. 7

Expression analysis of MsSQE1 in the indicated alfalfa tissues and under different conditions A: Analysis of the relative expression level of MsSQE1 in the indicated alfalfa tissues using qRT-PCR; B, C, D: Relative expression level of MsSQE1 in alfalfa seedlings treated with UV, GA3 or ABA, * and ** indicate P＜0.05 and P＜0.01, respectively (Student’s t-test). The same as below"

Fig. 8

The relative expression level of MsSQE1 (A) and the change of saponin concertation(B) in different tissues induced by MeJA in alfalfa"

Fig. 9

Identification of transgenic alfalfa and effect of overexpression MsSQE1 on total saponin content of alfalfa A: Verification of positive transgenic plants; B: Analysis of the relative expression level of MsSQS by qRT-PCR in the three representative transgenic lines; C: Content of total saponins in the indicated transgenic lines. CK+: Plasmid harboring 35S::MsSQS (positive control); CK-: pBI-121 empty vector (negative control); 1-10: Regenerated alfalfa plants resistant to kanamycin; 1,9,10: Three independent transgenic alfalfa lines. Bars represent the mean ± SD of three biological replicates"