Increasing Virulence Of Beauveria Bassiana By The Insect Chitinase (Bmchi) From Bombyx Mori

Entomopathogenic fungi is a kind of insect pest pathogens, which are wildly used to control agricultural and forestry pests and vectors of microbial pathogens of men, and thus have been intensively studied to develop mycoinsecticides for integration into management systems for insect pests. However, as true pathogens, all fungal biocontrol agents inevitably experience a latent period for infection development in host hemocoel and always kill insects more slowly than chemical insecticides. These disadvantages have often discouraged the use of fungal formulations in insect pest control. It is necessary to explore more effective means to facilitating fungal action at acceptable inoculum levels. Genetic manipulation provides a promising approach to altering fungal physiology and infectivity, which can be done by integrating one or more virulence encoding genes into a fungal candidate. Unfortunately, only a few candidate genes are available for genetic improvement of pathogen, which were all from fungal pathogen. So it is necessary to find the insecticidal gene from other organism to improve the fungal pathogens.During infection, the fungal pathogens produce a series of cuticle-degrading enzyme such as chitinases, proteases and lipases which facilitate penetration through cuticle. In addition, insect hosts, the target of fungal pathogen, produce chitinase to degrade the older cuticle to finish exuviations in insect metamorphosis. It is also demonstrated that the insect chitinase was only expressed at specific stages, such as molting, pupation and emergence. And thus abnormality expressing chitinase would interference insect development, which suggested that insect chitinase could be exploited as biological agent to control pest and used to improve virulence of entomopathogenic fungi.Here, it needs to improve virulence of entomopathogenic fungi using a insect chitinase (Bmchi) from silkworm. To investigate enzyme activity, Bmchi and BmchiΔc (partial sequence of chitinase which was deleted the ChBD (chitin-binding domain) coding region) were expressed in yeast and enzymes were purified, respectively. Then these two genes under control of fungal constitutive promoter PgpdA were introduced into Beauveria bassiana to evaluate their potential in improvement of insect fungal pathogen.Chitin-binding domain influences the enzyme activity, the results are as followings: (1) The binding efficiency of Bmchi to powder chitin was reduced and the hydrolysis efficiency of Bmchi against the chitin was decreased by deletion of the ChBD. (2) Deletion the ChBD changed optimum reaction temperature and optimum reaction pH of chitinase. The optimum reaction parameter of Bmchi was pH of 8 and at 25℃, but the optimum reaction of BmchiΔc was observed at pH 7 and at 35℃. (3) Deletion ChBD decreased thermostability of chitinase. Incubated at 40℃for 15 and 30 minutes, the activity of Bmchi was higher 1.6 and 2.2 times than that of BmchiΔc, respectively. After incubation for 60 minutes at 50℃, the remnants activity of Bmchi and BmchiΔc were 25% and 14%, respectively.In order to investigate the effects of Bmchi and BmchiΔc on virulence of B. bassiana, the transformants overexpressing Bmchi and BmchiΔc were used for bioassay anainst Myzus persicae. The results indicated that the virulence of the pathogen was significantly increased by overexpression of Bmchi, but overexpression of BmchiΔc did not influence the virulence of the pathogen. At the concentration of 1×10⁷conidial/mL, the median lethal time (LT₅₀) of the fungal pathogen was reduced 13.4% by overexpression of Bmchi and 5.8% by overexpression of BmchiΔc. At 138 hours after inoculation, the median lethal concentration (LC50) of B.bassiana was reduced 76% by overexpression of Bmchi, but the LC₅₀ was increased a little by overexpression of BmchiΔc. Our results suggested that the virulence of B. bassiana could be increased by constitutive expression of silkworm chitinase gene and the chitin-binding domain of the chitinase was essential for virulence.