Prokaryotic Expression And Functional Analysis Of 5' Fragment Of PtCDD Gene From Populus Tomentosa Carr.

The process of wood formation includes differitiation of cambium cells into xylem cells,which invoves progammed cell death (PCD) of developing xylem cells. DNase is an important participant in this PCD process. Further study on the DNase will not only provide molecular evidence for the PCD process, to find out whether it is the same process as animals exhibt or has own character in the wood forming process, but also help to understand the molecular basis of wood forming as well as resolving the issue on the time relation between secondary wall forming and the PCD's initiation. Therefore this study will express in E. coli and further charaterize poplar DNase in vitro to elucidate the mechanism of PCD process, which is important to improve the quality of the wood.The full-length Ca²⁺-dependent DNase (PtCDD) gene was amplified from the cDNA prepared from cambium tissue of Populus tomentosa Carr.. The gene was ligated into pET-30b(+) and the prokaryotic expression vector pET-30b(+)-PtCDDI was constructed. Then the expressing vector was transformed into E. coli BL21 (DE3). Although varible environment conditions were tested, the target protein had not been obtained. It seems that the full-length protein may have negative effcts on the expression in the E. coli thus the expressed product could not be accumulated. To solve this problem, the structure of the PtCDD gene was analyzed and the catalytic region with a part of the Ca²⁺ binding region of the PtCDD gene was amplified. By lacking the C- terminus, the ability of the PtCDD binding to DNA are expected to be weakened and the frgment of PtCDD gene could be obtained by expressed in E. coli. The PtCDD gene functional region was also constucted into the prokaryotic expression vector pET-30b(+) and its expressing vector pET-30b(+)-PtCDDF was transformed into E. coli BL21(DE3). The expressing condition had been optimizedand the obtained fusion protein was found mainly in the pellet in the form of inclusion body. Then the PtCDD protein was purified from the total protein with the TALON Affinity Column and it exhibted the ability to bind weakly to and digest DNAs. These results indicate that the truncated PtCDD can be expressed in E. coli (DE3) successfully, which could be speculated that the full-length PtCDD, as Dnase I, has very strong ability to bind and digest DNA, DNAs from both E. coli cells and the expression vectors were likely to be digested in E. coli thus could not be expressed in a greater amount. This study could support to the further research on character of the PtCDD gene, to raise its antibody to locate its distribution, for instance, in its role in PCD during wood formation.