Cloning, characterization, and expression of LeEIL - 1 , an Arabidopsis EIN3 homolog, in Lithospermum erythrorhizon

Ethylene is a crucial signal in regulating the biosynthesis of shikonin in Lithospermum erythrorhizon. Arabidopsis ethylene-insensitive 3 (EIN3) is the key transcriptional factor of the ethylene signal transduction pathway; thus, EIN3 homologs might play an important role in shikonin formation. Here, LeEIL-1, a gene with high similarity to EIN3, was cloned from L. erythrorhizon using a combination method of touch-down PCR and rapid amplification of cDNA ends. The full-length cDNA of LeEIL-1 is 2,359 bp, encoding a polypeptide of 635 amino acids. By inserting the entire coding region of LeEIL-1 into the pET32a (+) expression vector, the prokaryotic expression of LeEIL-1 was successfully induced by isopropyl β-d-1-thiogalactopyranoside in BL21(DE3)pLysS cells. Moreover, the recombinant LeEIL-1 protein was verified by western blotting with the Arabidopsis anti-EIN3 antibody. Real-time PCR results show that the mRNA level of LeEIL-1 was first dramatically induced within 3 h when the L. erythrorhizon cells were transferred from B5 to M9 medium for shikonin formation; subsequently, the transcripts of LeEIL-1 decreased to a relatively stable level. Tissue-specific expression analysis indicates that less LeEIL-1 mRNA accumulated in the stem and leaf than in the root, where shikonin was biosynthesized. These results imply that LeEIL-1 could be a possible reverse genetic target for revealing the relationship between ethylene and shikonin formation.