Bai Zhi-Wei, Pu Li-Mei, Tang Bi-Feng, Wang Qian, Cui Xiu-Ming, Liu Di-Qiu
Key Laboratory of Panax notoginseng Resources Sustainable Development and Utilization, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China.
Zhongguo Zhong Yao Za Zhi. 2018 May;43(9):1832-1837. doi: 10.19540/j.cnki.cjcmm.20180201.012.
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.
几丁质酶是一种将几丁质水解为N-乙酰葡糖胺的糖苷酶，广泛存在于植物细胞中，是植物抗真菌防御系统的重要组成部分。本文对三七几丁质酶基因PnCHI1的功能进行了表征。构建了PnCHI1的表达载体并在洋葱表皮细胞中瞬时表达，激光扫描共聚焦显微镜显示PnCHI1定位于细胞壁。还构建了PnCHI1的原核表达载体，并诱导纯化了PnCHI1的重组蛋白。体外抗菌试验表明，重组PnCHI1蛋白对茄形镰刀菌、尖孢镰刀菌和轮枝镰孢菌的菌丝生长具有较强的抑制活性。通过反向遗传学进一步验证了PnCHI1的功能。利用农杆菌介导法将PnCHI1表达载体转入烟草，通过qRT-PCR确认了PnCHI1的表达。通过叶片接种实验发现，转基因烟草对茄形镰刀菌的抗性显著提高。结论如下：PnCHI1是一种定位于细胞壁的几丁质酶，可抑制几种引起三七根腐病的真菌。该几丁质酶基因在烟草中的过表达大大提高了对茄形镰刀菌的抗性。PnCHI1可能是三七中参与防御根腐病的一个重要抗性基因。
