[Cloning and expression of the cry1Ac-tchiB fusion gene from Bacillus thuringinesis and Tobacco and its insecticidal synergistic effect].
A new fusion gene cry1Ac-tchiB was constructed to enhance the toxicity of crystal proteins, the cry1Ac gene of Bacillus thuringiensis strain 4.0718 was combined with a tchiB (deleted signal peptide and Enterokinase site sequence). In this process, the Enterokinase site sequence was inserted into the midst of these two genes. Then the combined fragment carrying the upstream promoter region and the downstream terminator region of cry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzyme digestions and subclonings two new expression vector pHUAccB6 and pHUAccB7 were obtained. The two vectors were transformed into B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant strain HAccB6 and HAccB7. The fusion gene was expressed and the expression product was detected by SDS-PAGE. The result indicated that the recombinant Cry1Ac-tchiB protein accumulated to the level of 61.38% of total bacterial proteins. Cry1Ac protein accumulated to the level of 42% of total bacterial proteins. Chitinase activities is 5.2 time more than that of the control strain. Under Atomic Force Microscopy and SEM of crystals protein, there were some bipy ramidal crystals with a size of 1.5 x 3.0 microm. Bioassay showed that the fusion crystals from recombinant strain HAccB6 and HAccB7 were high toxic against third-instar larvae of Helicourpa armigora with the LC50 (after 72h) value of 9.10 micro/mL and 11.34 micro/mL.The constructed fusion proteins had more toxicity than Cry1Ac crystal proteins. The study will enhances the toxicity of B. thuringiensis Cry toxins protein and makes a ground for constructing the fusion genes of B. thuringiensis cry gene and other foreign toxin genes and the recombinant strain with high toxicity.
Ding
Xue-Zhi
XZ
College of Life Sciences of Hunan Normal University, Key Laboratory for Molecular Biology of Microorganism of Hunan province, Changsha 410081, China. dxz963@tom.com
Luo
Zhao-Hui
ZH
Xia
Li-Qiu
LQ
Gao
Bi-Da
BD
Sun
Yun-Jun
YJ
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics*; D015153:Blotting, Western; D002688:Chitinases / Q000235:genetics*; D003001:Cloning, Molecular; D004357:Drug Synergism; D004731:Endotoxins / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics*; D007306:Insecticides / Q000494:pharmacology; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D014026:Nicotiana / Q000201:enzymology*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D002688:Chitinases