蜡梅几丁质酶基因的克隆与原核表达

在构建好蜡梅花cDNA文库并进行EST分析基础上,通过随机克隆测序,得到1个蜡梅几丁质酶的cDNA基因,命名为Cpchia(GenBank登录号:FJ749130).Cpchia cDNA全长为1 184 bp,开放阅读框为954 bp,编码317个氨基酸,其结构包括信号肽、几丁质结合域、可变交联区、催化区,无C端延伸区,为Class Ⅰ b型胞外几丁质酶,属于几丁质酶第19家族.将Cpchia克隆到原核表达载体pET-28a(+),在大肠杆菌BL21细胞中以包涵体形式表达融合蛋白,利用透析法获得复性蛋白,其几丁质酶活性经DNS法检测达到200 U·mL~(-1).酶活性和稳定性分析表明,试验条件下,pH 7.0有利于酶的稳定和活性发挥,40 ℃活性最高,在0 ℃低温下也有较高活性.上述结果说明,分离的Cpchia基因编码蛋白具有几丁质酶活性,而且可能与蜡梅花的抗寒性形成有关.

Cloning and Prokaryotic Expression of a Chitinase cDNA from Chimonanthus praecox

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 Ⅰ b 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 BL21 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.