Cloning, Expression And Insecticidal Activity Of Novel Cry2 Genes From Bacillus Thuringiensis

In this study, the novel types of cry2 genes in two Bt strains were studied. The SBT2 strain is a new Bt strain which is abstracted from the soil sample of Mt. Yuquan Hainan Province, containing cry2Ad gene. However the activity of cry2Ad has not been reported so far. The SC6H8 strain is one Bt strain abstracted from soil sample of Mt. Wuyi Fujian Province. The results of bioassay showed that this strain has the obvious body weight inhibitory action to the Bt-susceptible and CrylAc-resistant cotton bollworm (Heliothis avmigera), indicating that the insecticidal gene contained in the strain may not have cross-resistance to cry1Ac gene, which made it clear that the cry2 gene is included in the strain after it being identified. The cloning, expression and the activity of the two novel cry2 genes were studied.The SBT2 strain was analysed separately on three aspects: the crystal, plasmid profiles and protein molecular weight. The results showed that it produced large or small bipyramid crystal protein which the molecular weight was approximately 130kDa. The plasmid bands were similar to those large plasmids while different from small plasmids, compared with the B. thuringiensis subsp.kurstaki HD-1. The results of PCR-RFLP revealed that the strain contains cry1Aa, cry1Da, cry1Hb, cry1Jb, cry1Ka, cry1Ib, cry2Ad gene. A novel cry2Ad gene was cloned and sequenced from the strain. The result of BLAST in NCBI showed the similarity between Cry2Ad and Cry2Ad1 is 99%. The cloned gene is the fourth level cry genes. The cry2Ad gene was expressed by pEB, the high-level prokaryotic expression vector. The bioassay results showed that the Cry2Ad showed lower activity against cotton bollworm, corn borer (Ostrinia furnacalis), diamondback moth (Plutella xylostella) and gypsy moth (Lymantria dispar), and have no activity to the Phaedon brassicae (Colaphellus bowringi).The crystal, plasmid profiles, protein molecular weight analysis were performed to wild strain SC6H8. The results demonstrated that SC6H8 produced small bipyramid shape crystal protein expressing 130kDa and 60kDa, whose plasmids was much richer than HD-1. The results of PCR-RFLP showed that the strains contains cry1Aa, cry1Ba, cry1La, cry1Ia, cry2Aa, cry2Ab, novel cry2 gene. The novel cry2 gene was cloned. The protein BLAST results revealed that the Cry2Ax which belongs to the group of third level of holo-type gene has the highest homology with the Cry2Ab8 (93%). The cry2Ax gene was expressed by pET21b expression vector. The bioassay results showed that the Cry2Ax had a better inhibitory effect on body weight of cotton bollworm and corn borer. The IC50 s is 8.70ug/ml and 9.23ug/ml, respectively. The IC50 of CrylAc-resistant cotton bollworm was 13.96ug/ml. Cry2Ax showed no activity against diamondback moth and Phaedon brassicae.Cry2Ax is the first cry2 holo-type gene in our country. The cry2Ax had three structure territory which was same to the known Cry2A protein, which was revealed by the analysis of the three-dimensional structure. The carbon skeleton of Cry2Ab and Cry2Ax was almost same, except the difference on the 355. Use overlaps PCR to obtain Cry2Ax two fixed-point mutants: Cry2Axv354-sp and Cry2Axv354-vp. Those two genes were expressed by pET21b expression vector. Cry2Axv354-sp and Cry2Axv354-vp have no activity to the cabbage moth. The IC50 of the two mutants to cotton bollworm were 1.632 ug/ml and 5.683ug/ml, respectively.