Studies On The Fusion Expression And Function Of Cry1Ac Gene From Bacillus Thuringiensis 4.0718 With Proteinase Inhibitor Gene Hwtx-11 From Spider

Bacillus thuringiensis is one of the most widely used insecticidal microbes. There were some shortcomings for all traditional Bt pesticides, such as narrow host range, low toxicity against target pest, increased resistance from insect. Therefore, it is required to improve the toxicity of recombinant strain by modern molecular biotechnology. In order to construct the recombinant strain with higher toxicity and broader insecticidal spectrum, this work constructs the fusion gene of cry1Ac and hwtx-11 about 4.3 kb, then studies its function.1. Construct of cry1Ac gene fused with hwtx-11The proteinase inhibitor hwtx-11 gene from spider behind an enterokine kinase sequence was synthesized using B. thuringiensis preference codons for the toxin' s amino acid sequence. The synthesized sequence was phosphorylated, annealed and ligated with the pUAc digested with Bg1â…¡and Munâ… , which harbored cry1Ac gene, and produced the intermediate vector pUA11. Then the fusion gene with cry1Ac and hwtx-11 was obtained by digestion of pUA11 with Sal I and BamH I and cloned into the shuttle vector pHT315, produced expression vector pHA11, which was electroporated into the B. thuringiensis acrystalliferous strain Cry^-B and produced recombinant strain BHA11. The results of SDS-PAGE and Western blot showed that BHA11 produced 136.6 kDa fusion proteins. Bioassays indicated that the LC₆₀ against S. exigua Hubner and H. armigera larvae of BHA11 were 12.3μg/ml,23.7μg/ml, the value of LC₅₀ of BHAc against the two insects were 19.1μg/ml, 30.6μg/ml. The toxicity of BHA11 against S. exigua Hubner and H. armigera larvae was 1.6 times,1.3 times than that of BHAc, respectively.2. Effects of truncated Cry1Ac C-terminal to the fusion expressionThe pHA11 digested with Xhoâ… and Bglâ…¡enzymes, and the ends was blunted by T4 DNA polymerase and Mung Bean Nuclease respectively, then ligated and produced truncated Cry1Ac C-terminal plasmid pHA11T. The expression vector pHA11T was electroporated into B. thuringiensis acrystalliferous strain Cry^-B, then produced recombinant strain BHA11T. SDS-PAGE and Western blot showed that BHA11T produced 74.3 kDa fusion proteins. Bioassays indicated that the crystal proteins produced by BHA11T were still functional in killing the S. exigua Hubner and H. armigera larvae, the value of LC₅₀ were 34.7μg/ml,63.7μg/ml, and lower 0.8 times,1.1 times than that of BHAc, respectively.3. Synergism between HWTX-11 and Cry1Ac The spider toxin gene hwtx-11 has been synthesized by chemical method and cloned into the prokaryotic vector pGEX4T-1. The HWTX-11 proteins were expressed by IPTG induction and detected by SDS-PAGE and Western blot with polyclonal antibody. The results showed that the molecular mass of the fusion protein was 33 kDa. Bioassay showed that the value of LC₅₀ against S. exigua Hubner and H. armigera larvae reached 86.6μg/ml,119.4μg/ml, respectively. Moreover, it also showed synergistic effects were present between GST-HWTX-11 and Cry1Ac against S. exigua Hubner and H. armigera larvae.In order to effectively express the fusion genes of cry1Ac and hwtx-11 in Bt, the study optimized a series of methods on the strategy of the experimental design. Firstly, the dual overlapping promoter of Btâ… and Btâ…¡carried by cry1Ac gene itself, which is the most prevalent promoter present in Bt and also suitable to express the fusion gene in the acrystalliferous Cry^-B, was used to promote the expression of the fusion protein. Secondly, we kept the stem-loop structure and poly(T) structure in the downstream of the terminator of cry1Ac gene, which can effectively terminate the transcription and make mRNA stable in the process of translation. Thirdly, we synthesized the hwtx-11 gene using B. thuringiensis preference codons for the amino acid sequence to express the hwtx-11 gene from eukaryon in Bt and inserted the recognized sequence of enterokinase in order to proteolysed the fusion protein into active toxins in the middle gut of insect. Moreover, the effects of the CrylAc C-terminal to the expression of the fusion protein were also studied. The recombinant strain BHA11T constructed by this study had a lower toxicity than the control, may because the fusion proteins lose the C-terminal contained the rich cysteines, which can form the disulphide group and facilitate the formation of crystal bodies, and can not form the crystal bodies and degraded very easily in host after expression. Then we also have studied the synergism between the HWTX-11 and the CrylAc. The results of bioassays showed the HWTX-11 have good synergistic effects to Cry1Ac protein, maybe the different mechanism enhanced the activity against the insect greatly. It is important foundation for constructing the fusion genes with Bt cry genes and other foreign toxin genes, as well as constructing the engineering strains withhigher toxicity.