Identification, Molecular Cloning, and Recombinant Gene Expression of an Extracellular A-Amylase from Dactylis Glomerata L. Embryogenic Suspension Cultures

ABSTRACT

Somatic embryogenesis is a unique phenomenon in the plant kingdom and involves the transition of somatic cells into cells that are capable of forming an embryo. Proteins secreted into the medium of suspension cell cultures play an important role by either promoting or inhibiting embryo development. A monoclonal antibody MAb 3G2 raised against extracellular proteins from orchardgrass (Dactylis glomerata L.) embryogenic suspension culture recognized specifically a 48 kD protein with pI 5.2 (designated EP48) from the cell wall and culture medium of microclusters, which could serve as an early marker for embryogenic potential in D. glomerata cultures [Tchorbadjieva M., Kalmukova R., Pantchev I., Kyurkchiev S. 2005. Planta, 222, 811–819]. In this study, in-gel trypsin digestion of EP48 separated by 2-D gel electrophoresis and LC-MS/MS mass spectrometry identified the protein as α-amylase. The full-length cDNA encoding D. glomerata L. α-amylase (designated DgAmy1, GenBank accession number GU 067465.1) was cloned by rapid amplification of cDNA ends (RACE) and was shown to contain an open reading frame of 1284 bp encoding α-amylase protein of 427 amino acids with a calculated molecular mass of the mature protein of 44.742 kDa and pI of 5.12. Comparative analysis showed that DgAmy1 shares an overall identity of 59–89% with α-amylases from other plant species, and is most closely related to that of barley (Hordeum vulgare). Homology modeling using barley α-amylase Amy2 as a template revealed that DgAmy1 contains three domains (A, B, C) and active sites that are common with other plant α-amylases. However, it lacks the substrate binding sites 1 and 2 typical for cereal α-amylases. The recombinant DgAmy1 expressed in E. coli BL21 (DE3) and purified by Ni²⁺ Sepharose was biologically active as revealed by activity zymography using soluble starch as a substrate. The high yield (200 mg protein from 1 L culture medium) and purity (99%) of the recombinant DgAmy1 will be helpful for further understanding of the structure/function correlations and especially the role that this extracellular α-amylase plays at early stages of somatic embryogenesis.

Keywords:

• cell wall α-amylase
• Dactylis glomerata L.
• heterologous expression
• plant extracellular proteins
• RACE
• somatic embryogenesis