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- Title
Cloning, characterization, and expression of LeEIL- 1, an Arabidopsis EIN3 homolog, in Lithospermum erythrorhizon.
- Authors
Ailan Zou; Wenju Zhang; Qinyan Pan; Simei Zhu; Jingjing Yin; Runan Tian; Hongwei Gu; Xiaoming Wang; Jinliang Qi; Yonghua Yang
- Abstract
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.
- Subjects
MOLECULAR cloning; GENE expression in plants; ARABIDOPSIS; ETHYLENE; LITHOSPERMUM; HOMOLOGY (Biology); POLYPEPTIDES; PLANT genetics; POLYMERASE chain reaction; WESTERN immunoblotting
- Publication
Plant Cell, Tissue & Organ Culture, 2011, Vol 106, Issue 1, p71
- ISSN
0167-6857
- Publication type
Article
- DOI
10.1007/s11240-010-9895-1