RESUMO
In the study, specific primers were designed based on the CO Ⅰ gene sequence of Polyrhachis dives. By optimizing the genomic DNA extraction method and amplification conditions, we established an efficient, specific, and accurate DNA molecular identification method for Polyrhachis dives. In this method, the length of the target fragment was 294-308 bp, and the other counterfeits had no target bands. In this paper, the specific identification method of the origin of Polyrhachis dives established can be used to identify the medicinal materials of Polyrhachis dives accurately.
RESUMO
The proteins encoded by the Brassica juncea chitinase gene BjCHI1 and its derived genes BjCHI2 and BjCHI3 were expressed by Multi-copy Pichia expression system. The chitinase activity of FPLC purified BjCHI1, BjCHI2 and BjCHI3 were tested and the results showed that all the three proteins degraded both CM-chitin-RBV and colloidal chitin. The Km values of BjCHI1, BjCHI2 and BjCHI3 for CM-chitin-RBV were estimated as 0.799 mg/mL, 0.544 mg/mL and 0.793 mg/mL, respectively. When the colloidal chitin was used as substrate, the Km values were 0.281 mg/mL, 0.388 mg/mL and 1.643 mg/mL, respectively, indicating chitin-binding domain can increase affinity of chitinase to insoluble substrate. In the agglutination activity assay, only BjCHI1 shows activity when the protein concentration was more than 33 micrograms/mL, while BjCHI2 and BjCHI3 without agglutination activity even when the concentration was increased as high as 800 micrograms/mL. This means that the two chitin-binding domains in BjCHI1 are essential for agglutination and BjCHI1 is the first protein which shows both chitinase and agglutination activity identified so far in plants.