The Cloning And Characterization Of CDNA For ECBP21 From Angelica Dahurica

In recent years, Ca²⁺ signal system (Ca²⁺ , CaM, and CaMBPs) has been thoroughly researched. Many intracellular CaMBPs have been detected and studied. In our laboratory, a series of experiments has provided evidence that the presence of CaM in the apoplast area is universal, and extracellular calmodulin plays an important role in the plant growth and development as a polypeptide signal. This has become one of the latest progresses in plant CaM research work.In the process of study for the extracellular CaM, the research in our lab indicates that CaMBPs also possibly exist in cell wall area. For the first time, an extracellular Ca²⁺-dependent CaMBP (named: ECBP21) was purified from 0.1mol/L CaCl₂ extracts of Angelica dahurica suspension-cultured cells (Tang et al. 1996). Furthermore, our preliminary evidence shows that the ECBP21 protein influences on proliferation of this cultured cell (Mao et al. 1999). By using methods of biochemistry and physiology, the above experiments only provided preliminary evidence for the existence and possible functions of the extracellular CaMBPs. In order to prove the real existence of the extracellular ECBP21 and its functions by means of molecular biology, we first cloned and identified the full-length cDNA for an extracellular CaMBP (ECBP21).The purified protein was electrobloted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5 "RACE cloning. The cDNA contains 947 nucleotides that codes for a precursor protein of 216 amino acids. Sequence analysis shows that the N-terminal 1-25 amino acid sequence is a predicted signal peptide and other 26-216 amino acid sequence is a mature peptide which has no transmembrane domain. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E.coli BL21(DE3). A protein with relative molecular mass 21kD was expressed in E.coli. Using a biotinylated-CaM gel overlay technique, the expression protein has the ability to bind CaM in the presence of Ca²⁺. Theseresults further defined ECBP21is a Ca²⁺-dependent CaM-binding protein. Following researches verified that the CaM-binding domain exists in the C-terminal of ECBP21. Southern blot analysis shows that there are two ECBP21 gene copies in genome.Second, The ECBP21 was localized in the cell wall area by immuno-gold electron microscopy and by using GFP as a reporter. The results indicted that ECBP21 is a kind of secretive protein and ECBP21 is an apoplast polypeptide. These experiments provided the first and direct evidence for the existence of the extracellular CaMBPs.Third, we examined the expression of ECBP21 dynamics during the incubation of Angelica suspension-cultured cells and stress treatments through Northern blot analysis. During the incubation of Angelica suspension-cultured cells, the expression of ECBP21 gene increased from 0 to 12th day, and it reached a peak on the twelfth day. While the expression of CaM gene increased from 0 to 9th day, and it reached a peak during the 6th-9th day. These results indicated that ECBP21 may involve in the regulation of cell growth. The stress treatments (such as: heat shock, osmotic and salt stresses) and JA (100uM) treatment repressed the expression of ECBP21 gene. They indicated that ECBP21 may have some relative with the response to stress treatments.Further more, we constructed the binary vector containing ECBP21-GFP chimeric gene and got transgenic plants by using the Agrobacterium vacuum infiltration method. The phenotype observation is still in progress.Thought the above studies, we first cloned and identified an extracellular CaMBP (ECBP21) cDNA, and provided direct evidence for the existence of the extracellular CaMBPs. This work laid a foundation for elucidating the functions of ECBP21 by means of molecular biology. Further studies about ECBP21 may be benefit to elucidating the mechanism of apoplast calmodulin o...