Abstract:

A chitinase gene was isolated based on the library construction from Chimonanthus praecox flower and its EST analysis, and was named as Cpchia (GenBank accession No. FJ749130). The full length of Cpchia sequence was 1 184 bp, containing an opening reading frame of 954　bp. It encoded a polypeptide of 317 amino acid residues including signal peptide, cysteine-rich domain, hinge region, and catalytic domain. The homogeneity in structure and sequence showed that this cDNA would belong to Class Ⅰb chitinase, a member of the 19th chitinase family. We inserted this gene into the prokaryotic expression vector of pET-28a(+) and it expressed in Escherichia coli BL2l as inclusion bodies. The refolded proteins were obtained after stepwise dialysis, and the highest activity was 200 U·mL^-1 measured with 3,5-dinitrosalicylic acid (DNS) method. The enzyme was stable at pH 7.0， and had the most activity at 40 ℃ and remained active at 0 ℃. The results showed that Cpchia encoded the protein with chitinase activity and probably was related to cold tolerance of Chimonanthus praecox blooming.

Key words: chitinase, cloning, prokaryotic expression, Chimonanthus praecox