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GS115-pPIC9k-ChiA4.0 Fermentation HaSNPV chitinase production separation and extraction , insect antibacterial mechanisms and prevention research field

Author: XiaTian
Tutor: HuiYouWei
School: Northwestern University
Course: Biochemical Engineering
Keywords: HaSNPV chitinase purification toxicology field test
CLC: TQ925
Type: Master's thesis
Year: 2011
Downloads: 57
Quote: 0
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Abstract


Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) express extracellular chitinase, which hydrolysis chitin in cell wall of insect and fungi specifically, in order to inhibit insect and fungi, even make host inactivation. The gene-engineered strain GS115-pPIC9k-ChiA 4.0 which express chitinase has been constructed successfully by constructing chitinase gene from HaSNPV into vector pPIC9k and transferring into host Pichia pastoris GS115. Further screened, induced and optimized fermentation conditions of multi-copy strains, obtained soluble HaSNPV chitinase. Purification of chitinase, researched the mechanism of insecticidal and antibacterial, analyzed the control effect of chitinase on pest in field are main contents of this study.The supernatant of fermentation was salted out with (NH4)2SO4, the precipitation in 60%-75% saturation was collected. Then purified the precipitation by DEAE-Sepharose Fast Flow anion exchange chromatography which was eluted with 0.4 mol/LNaCl in buffer (pH 8.0). The loading amount is 4.0 ml, and the velocity of eluate is 0.5 ml/min. After that sample through Sephadex G-100 gel filtration chromatography. By SDS-PAGE, HaSNPV chitinase was electrophoresis pure. The enzymatic activity was 438.44 U/ml, the rate of protein recovery rate and purification fold were respectively 19.65% and 35.29.The corrected mortality of stomach toxicity was 75% when the chitinase concentration was 1.5 mg/ml at 72 h, the contact toxicity was 40.74% under the same conditions. The result suggested that there were double function, the contact toxicity was weaker than stomach toxicity under the same conditions. Chitinase had no antifeedant on plutella xylostella.The antagonism of HaSNPV chitinase on six common agricultural pathogens were different, the relative inhibition rate of chitinase on Helminthosporium sativum was 86.05%.The field test of efficacy showed that common chemical pesticides can control plutella xylostella larvae, but long-term use is bound to cause drug resistance and serious damage of the ecological balance. HaSNPV chitinase has no antifeedant effect on plutella xylostella. It can control pest for a long time,9 days later, the control effect is 93%.The research of cell structure in plutella xylostella after treatment suggested that HaSNPV chitinase can hydrolysis chitin of epidermis, midgut and peritrophic membrane, then destroyed cells, lead to pest death.

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