The Vegetative Insecticidal Protein Genes Of Bacillus Thuringiensis And Expression In Transgenic Rice

Bacillus thuringiensis(Bt)has been extensively explored for biological control of insect pests.Vip3 are insecticidal proteins secreted during Bt vegetative growth stage,and share no significant similarity with any known Bt crystal toxins in amino acid seuqence.This thesis is focused on the discovery and creation of novel Vip3 toxins and their utilization in transgenic rice for insect resistance.To identify new vips genes,a large number of Bt isolates were screened by PCR using two pairs of primers that are enable to amplify DNA fragments from vip3Aa1 gene.A minilibrary was generated by using plasmids from Bt isolate LG13,which carrys a novel vip3 gene.The 5' end and 3' end of the novel vip3 gene was identified in the minilibrary.The full-length novel vip3 gene was then obtained by PCR,and named vip3Ab2.The E.coli expressed protein of this gene showed high insecticidal activity against fall armyworm,Spodoptera frugiperda,and corn earworm, Helioverpa zea,but very low activity against the silkworm,Bombyx mori.The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin,Vip3Aa1.Vip3AbAa,consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1,and Vip3AaAb,consisting of the N-terminal region of Vip3Aa1 and the C-terminal region of Vip3Ab2,were obtained.The chimeric Vip3AaAb had decreased toxicities to insect bioassyed,but the chimeric Vip3AbAa had inecreased toxicities to fall armyworm,silkworm and cabbage looper. While both Vip3Aa1 and Vip3Ab2 showed no detectable toxicity against the European corn borer,Ostrinia nubilalis,the chimeric protein Vip3AbAa became insecticidal to European corn borer.Furthermore,both Vip3Aa1,Vip3Ab2,and Vip3AbAa were highly insecticidal to a strain of cabbage looper,Trichoplusia ni,that was highly resistant to the Bt endotoxin Cry1 Ac,thus experimentally showing for the first time the lack of cross-resistance between Bt Cry1 A proteins and Vip3A toxins. The results in this study demonstrated that vip3Aa1,vip3Ab2 and their chimeric vip3 genes could be excellent candidates for engineering a new generation of transgenic plants for insect pest control. For better mangenment of insect resistance against Bt toxins,a crystal toxin and a Vip3 toxin were used for creation transgenic rice.A fusion protein of Cry1Ab and Vip3H was created and named as Cry1AbVip3H.When treated with trypsin,this fusion protein expressed in Escherichia coli strain BL21(DE3)released the cores of both Cry1ab and Vip3H,which were the same as protein cores genarated by trypsin proteolysis of the individual Vip3H and Cry1AbVip3H.Activity assays indicated that the E.coli expressed Cry1AbVip3H was highly insecticial to fall armyworm,S. frugiperda,black cutworm,Agrotis ipsilon,corn earworm,H.zea,cotton bollworm,H. armigera,and tobacco budworm.Heliothis virescens.The T-DNA construct containing the cry1Abvip3H gene under the control of a maize ubiquitin promoter was transformed into rice.Western bolt analysis showed the presence of Cry1AbVip3H in the all parts of transgenic rice plants.Transgenic rice expressing Cry1AbVip3H significant reduced leaf damage caused by rice case worm,Cnaphalocrocis medialis.