| Overexpressing chitinases in transgenic plants is one of the hot points in plant genetic engineering for fungal resistance. Based on the occurrence of an N-terminal chitin-binding domain and on amino acid sequence homology at the catalytic domain, plant chitinases can be classified into nine classes. Chia6 chitinase has half a chitin-binding domain, Chial, Chia4, Chibl, Chid and Chidl chitinases have one such domain while Chia5 premature chitinase has two chitin-binding domains. But BjCHIl gene from Brassica juncea encodes the chitinase with two chitin-binding domains that is different from ChiaS. Studying on the characters of this gene and its protein is our goal in this research, especially focus on roles of the two chitin-binding domains.In order to express BjCHIl, Escherichia coli expression system was previously used, but not succeeded due to its detriment to bacteria. In this study, Pichia pastoris expression system was used to express BjCHIl. According to the sequence of BjCHIl cDNA and the multiple cloning sites, three special primers were designed and synthesized. After PCR with the primers and templates, the fragments of BjCHIl encoding the mature protein, BjCHI2 encoding the protein with one chitin-binding domain and BjCHB encoding the protein without chitin-binding domain were amplified, respectively. The amplified fragments and pPIC9k were digested by EcoR. l+Not I, mixed and ligated by T4DNA ligase, thus expect DNA fragements were inserted into the EcoR l+Not I site of pPIC9K, called yeast expression plasmids pP17, pP28 and pCat, respectively. Then the three vectors were transferred into yeast KM71 and experiments were carried out to select strains that could highly express the genes by secreting the proteins, BjCHIl, BJCHI2 and BjCHB which contain two, one and zero chitin-binding domain, respectively. The optimal inducing time of the three recombinants yeast strains was 2-3 days. Western blotting was carried out to confirm the proteins. After scale-up expression, FPLC was used to purify the three proteins that was suitable for the following experiments.Enzymatic characteristics of the three proteins were studied. The results showed that all of them have chitinase catalytic activity on soluble substrate (CM-chitin-RBV) and insoluble substrate (colloidal chitin), indicating that the chitin-binding domain is not essential for catalytic activity. When CM-chitin-RBV was used as the substrate, three proteins was stable at pH 3-10 and below 70℃; the optimal temperature was 50℃; the optimal pH of BjCHIl was pH6, while that of BJCHI2 and BjCHB was pH5. The Km of BjCHIl, BJCHI2 and BjCHB for CM-chitin-RBV were 0.799 mg/ml, 0.544 mg/ml, 0.793mg/ml, respectively, with little difference among them, indicating chitin-binding domain didn't affect Km for solution substrate. When collidal chitin as the substrate,BjCHIl and BJCHI2 were stable below 60℃ and at pH3-10 but BjCHD at pH3-9 and below 60℃; the optimal temperature of BjCHIl was 60℃ while that of BJCHI2 and BJCHI3 was 40℃; the optimal pH of three proteins was pH5 and pH9. The Km of BjCHIl, BJCHI2 and BjCHD for collidal chitin were 0.281 mg/ml, 0.388mg/ml, 1.643mg/ml, respectively. The Km of BjCHD was 5.8 and 4.2 times of that of BJCHI2 and BjCHD, respectively. This means chitin-binding domain take a more important role in degrading insoluble substrate such as colloidal chitin. The chitinase activity of three proteins on both soluble and insoluble substrate was inhibited by Fe3+, Cu2+and Hg2+.The results of agglutination assay showed that only BjCHIl showed agglutination activity when the protein concentration was more than 33ug /ml. Therefore, BjCHIl is the first protein which shows both chitinase and agglutination activity identified so far in plants. While BJCHI2 and BjCHD showed no agglutination activity even when the concentration was increased as high as 800ng/ml, indicating that the two chitin-binding domains in BjCHIl were essential for agglutination. BjCHIl had stronger inhibition activity on Bacillus megaterium than that of BJC...
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