Purification Of An Elicitor, Cloning And Prokaryotic Expression Of An Elicitin Encoding Gene

Elicitors are molecules that can bind to the highly specific receptors in plant plasma membrane and then induce defense response of the plants. The elicitors from mycelium cell wall or culture filtrate of some fungi are especially interesting to researchers, as they can be used to study the mechanisms of hypersensitive response (HR), and their encoding genes may be used to transform plants to get new plant materials with resistance to pathogens. This thesis is focused on the purification of an elicitor, cloning and prokaryotic expression pf an elicitin encoding gene in a Phytophthora isolate from Changchunhua leaves. The main results are as follows:1. A proteinaceous product was extracted and purified from the culture filtrate of this Phytophthora isolate through the procedures of boiling-water bath, ammonium sulfate precipitation, non-denaturing polyacrylamide gel electrophoresis (PAGE) and electroelution. It showed an apparent molecular weight of about 31kD in SDS-PAGE gel. Inoculation with this protein caused necrosis in tobacco leaves. Treating suspension cultured tobacco cells with this protein resulted in 44.6% cell death after 12hr, and 96.1% cell death after 24hr. Partial N-terminal sequence of this protein was sequenced and was found to be highly similar as those of some reported elicitors. As the molecular weight of this protein is obviously different from reported elicitors, this protein may be a novel elicitor.2. Internal transcribed spacers (ITS) of ribosomal DNA are highly conserved in different isolates of cetain fungus species, but have abundant variation between different fungus species, which is the basis of their wide application in phylogenetic research. Here an rDNA-ITS sequence of the Phytophthora isolate was cloned and compared with those of many identified Phytophthora speicies. As a result, its rDNA-ITS sequence was found to have 99% similary with those of Phytophthora nicotiana isolates. Thus the Phytophthora isolate used in this thesis is inferred to be Phytophthora nicotiana.3. Two 294bp sequences were amplified by RT-PCR with the RNA of the Phytophthora isolate using primers designed based on the elicitin encoding gene of Phytophthora nicotiana. One sequence was confirmed to a Phytophthora nicotiana elicitin encoding gene according to sequencing results, while the other sequence was the first sequence with mutations at the sites of 63 and 223. Theoreticaly, the mutation at the site of 223 can change the threonine residue into an alanine residue at the site of 75 in its encoded protein. Both sequences were cloned into pGEX-4T-l and expressed in E.coli (strain BL21). Both expressed proteins showed an apparent molecular weight of about 36kD, which is equal to the sum of molecular weights of GST and elicitin, and suggests that the two cloned sequences were successfully expressed in the form of fusion protein in E.coli under the induction of IPTG Both fusion proteins could induce hypersensitive response of tobacco leaves, and induce obvious death of suspension cultured tobacco cells after treatment for 12hr or 24hr. The biological activity of the mutated product was slightly higher than that of the non-mutated product.