关键词: 毛白杨, PtCDD基因, 原核表达, 功能鉴定, 融合蛋白

Abstract:

The development of secondary vascular system in woody plants is a complex process including cambial cell differentiation, cell expansion,secondary wall formation and final programmed cell death (PCD). In order to validate the role of a Ca²⁺ dependent DNase, found in the differentiating xylem, in the PCDprocess, it is necessary to express its gene of PtCDD and purify the product in a large quantity in order to raise antibody for further immunolocalization analysis. In this study, the 5′ fragment of 637 bp was amplified by PCR from the Populus tomentosa cambium cDNA gene with primers designed according to the PtCDD gene sequence published in GenBank, and the fragment was ligated into the vector PET 30b(+) to construct the prokaryotic expression vector for PtCDD gene. The expressed PtCDD (HIS) 6 fusion protein was purified and the function was analyzed. The results showed that the PtCDD fragment could be successfully expressed in the Escherichia coli cells and the fusion protein exhibited the DNase activity. This achievement provided a solution to the problem that the entire protein could not be expressed in E. coli probably due to its strong digestion activity on DNAs, so that a convenient method of the antibody preparation became available to further investigate the role of PtCDD in PCD.

Key words: Populus tomentosa, PtCDD gene, prokaryotic expression, function identification, fusion protein