毛白杨PtCDD基因5′片段的原核表达及功能分析(英文)

Prokaryotic Expression and Functional Analysis of 5′-Fragment of PtCDD Gene from Populus tomentosa

根据GenBank中毛白杨钙离子依赖型脱氧核糖核酸酶(PtCDD)基因序列,以毛白杨形成层区域的cDNA为模板,经PCR扩增出该基因上游637 bp的cDNA片段。将该片段与PET-30b(+)载体连接,构建杨树PtCDD基因片段原核表达载体并进行表达研究,获得大量的PtCDD-(HIS)6融合蛋白。并进一步对该蛋白进行纯化和功能检测。结果表明:PtCDD基因片段能够在大肠杆菌体内表达出具有DNase功能的蛋白,克服了因表达全酶可能强烈降解DNA所带来的不能最终获得表达产物的问题,为今后PtCDD蛋白的抗体制备以及进一步研究奠定了基础。

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 Ca^2＋ -dependent DNase, found in the differentiating xylem, in the PCD process, 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.