Cloning Of Novel Cry Gene From Bacillus Thuringiensis And Bioactivity Of Insecticidal Proteins

In the globe, the damage from Insects is one of the most important factors for loss ofagricltuaI productions yearly. Bacillus thuringiensis has been widely used as a microbialinsecticide because of plenty advantages including specific insecticidal activity, safety to humanand mammal, harmony to the environment. Transgenic plants to control insect pests have been atpractical stage, owing to the development of plant gene engineering. Therefore, insecticidalgenes of B. thuringiensis will be applied much more in future for various insect pestsbiocontrols.In this study, 10-kb DNA fragment containing novel cry gene was cloned by establishment ofplasmid DNA library of Bt C006 isolate. After subcloning and DNA sequencing analyses, twocry genes were revealed from this large fragment, one of them was the same as cry1Ab13 gene,another was novel, and its nucleic acid identity was low 80% compared with that of known cry1genes. The nucleic acid sequence of this new gene was registered in GenBank with the accessionnumber EF550989.cry1Gb2, the novel gene cloned in this lab previously was inserted into expression vectorspSTK and pET21 respectively, After transformation and inducement, cry1Gb2 gene could beexpressed normally with molecular mass of 133kDa in both Bt crystal mutant HD73^-and E. colistrain BL21, and typical bipyramidal crystal could be detected under microscopy. The bioassayresult indicated that Cry1Gb2 was highly toxic to larvae of Plutella xylostella, lethal concentration50% was 7.48μg/ml.Individual and combined protoxin proteins were performed respectively aiming at Chilosupperssalis, very important insects for rice. The insecticidal results for C. supperssalis larvaeshowed that LC₅₀ of Cry2Aa, Cry2Ab and Vip3Da was 27.59, 13.88 and 10.29μg/g, separately;and further bioassay results of combination between different Cry protoxins demonstrated that sixof them were remarkable, LC₅₀ of CrylAb +Cry2Aa, Cry1Ab+Cry2Ab, Cry1Ab+Vip3Da,Cry1Ca+Cry2Aa, Cry1Ca+Cry2Ab and Cry1Ca+Vip3Da was 0.39, 0.43, 0.15, 0.79, 0.64 and 3.39μg/g respectively.The lethal concentration 50% of Cry 1Ca, Vip3Aa, Vip3Af and Vip3Da against Sesamia inferenswas 4.28, 16.64, 57.54 and 13.93μg/g, respectively.All these results elucidated that known Bt toxin protein and their combination withnovel andintense pesticidal activities will be powerful candidates for inset pest biocontrol and resistant management.Cultural media were optimized in order to improve the quality and quantity of different Cryproteins. The beef medium containing 50 mM Tris-HCl with pH value 7.2 was optimum toharvest abundant intact protoxin crystal, and activity of alkaline proteases was inhibited,degradation of Cry proteins could be avoided as well.Rearing and bioassay metheds were established for longhorn beetle (Anoplophora nobilisGanglbauer), which is severe damage against forest yearly, meanwhile two isolates of Bacillusthuringiensis were screened with acivity against longhorn beetle larvae. All of this work will besignificant for biocontrol of this insect species.