ABSTRACT
Chitinases, a glycosidase enzyme that hydrolyzes chitin to N-acetylglucosamine, are widely found in plant cells, and they are an important part of plant antifungal defense system. The function of a Panax notoginseng chitinase gene PnCHI1 was characterized in this paper. Expression vector of PnCHI1 was constructed and transiently expressed in onion epidermal cells, and laser scanning confocal microscopy demonstrated that PnCHI1 was localized in the cell wall. Prokaryotic expression vector of PnCHI1 was also constructed, and recombinant protein of PnCHI1 was induced and purified. In vitro antibacterial assay showed that recombinant PnCHI1 protein had strong inhibitory activity on the mycelium growth of Fusarium solani, F. oxysporum and F. verticillioide. The function of PnCHI1 was further verified by reverse genetics. PnCHI1 expression vector was transferred into tobacco by Agrobacterium tumefaciens and expression of PnCHI1 was confirmed by qRT-PCR. It was found by leaf inoculation experiment that resistance of transgenic tobacco to F. solani was significantly increased. It is conclnded that: PnCHI1 is a chitinase localized in the cell wall, which inhibits several fungi which cause the root rot disease of P. notoginseng. Overexpression of this chitinase gene in tobacco greatly increased resistance to F. solani. PnCHI1 may be an important resistance gene in P. notoginseng that participates in the defense against root rot disease.
ABSTRACT
Objective To express recombinant human cardiotrophin 1 (huCT 1) protein with biological activity in E coli Methods huCT 1 open reading frame gene from plasmid pBSSK huCT1 was cloned into plasmid pMD18 T by PCR and T A cloning, and then cloned into prokaryotic GST fusion expression vector pGEX 2T to give pGEX2T huCT1 After pGEX2T huCT1 expression was induced by IPTG in E coli at different temperature and different time, the expression level and the proportion of the soluble protein were analyzed by SDA PAGE Then the soluble GST/huCT1 was purified by immobilized glutathione columns The GST fusion protein was cleaved by thrombin and purified again The recombinant huCT 1 with biological activity was identified according to the survival of axotomized sciatic motoneurons Results After the induction of pGEX2T huCT1 DH5? cells by IPTG at 29 ℃ for 4 h, the highest expression level of the recombinant protein was about 1/5 of total cell proteins, and the soluble portion was about 2/5 of fusion protein Purification of the soluble portion and thrombin cleaved fusion protein resulted in 85% and 80% purified recombinant GST fusion protein and huCT 1 protein, respectively Recombinant huCT 1 protected 55% motoneurons in spinal cord against sciatic axotomy in vivo in adult rats Conclusion Recombinant huCT 1 has biological activity in rat neurons