Prokaryotic expression, polyclonal antibody preparation of the stilbene synthase gene from grape berry and its different expression in fruit development and under heat acclimation

摘要

Stilbene synthase (STS, EC 2.3.1.95) leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. STS expression and regulation are important. Temperature is one of the main external factors affecting phytoalexin accumulation in plant tissues, the effect of temperature on resveratrol synthesis and stilbene synthase expression in grape berries has not been reported before. Here we cloned the full-length sts cDNA with 1179 bp from grape berry via PCR, and then introduced into an expressed plasmid pET-30a(+) vector at the EcoRI and Xhol restriction sites. With the isopropyl-beta-D-thiogalactoside (IPTG) induced, the pET-sts was highly expressed in Escherichia coli BL21 (DE3) pLysS cells. A fusion protein with the His-Tag was purified by Ni-NTA His . Bind Resin and then used as the antigen to immunize a New Zealand rabbit. Furthermore, the antiserum was precipitated by 50% saturated ammonium sulfate and DEAE-Sephadex A-50 chromatography to obtain the immunoglobulin C (IgG) fraction. These results provide a substantial basis for the further studies of the STS in grape berry as well as in other species of plants. The sts expression in fruit development and in response to heat acclimation was then assayed. The results indicated STS was regulated in fruits depending on the developmental stage and significantly accumulation of STS mRNA and synthesis of new STS protein during the early of heat acclimation, this work offers an important basis for further investigating the mechanism of post-harvest fruit adaptation to environmental stresses. (C) 2008 Elsevier Masson SAS. All rights reserved.