Toxic Fragment And Toxicity Improvement Of Nematicidal Proteins Cry55Aa And Cry5Ba From Bacillus Thuringiensis

Bacillus thuringiensis is a group of spore-and crystal-forming bacteria. And the crystal proteins exhibit great toxicity against many kinds of agricultural pests, including insect, nematode and mite. And the crystal proteins produced by Bacillus thuringiensis is thought as a potential tool in genetically modified plant breeding to control these pests. In the present study, the toxic fragments of nematicidal crystal proteins Cry55Aa and Cry5Ba were predicted and verified, as well as the improvement of toxicity of the crystal protein Cry5Ba. In addition, the study on Pasteuria penetans against plant root-knot nematode was also included in the graduation thesis as a partial work during my master period.Firstly, the toxic fragment of crystal protein Cry55Aa was identified in the present thesis. It has been proven that most of crystal proteins from B. thuringiensis were digested by host proteinase to a little core toxic fragment in which the partial N terminal amino acid residues and total C half of the proteins were cut off. So we assumed that the Cry55Aa might also have a toxic and nontoxic region. But by bioinformatical analysis we found that there are not related protein sequences in NCBI protein database, suggesting that the Cry55Aa might be a new protein family member. To verify whether the Cry55Aa has a toxic fragment, a series of truncated mutant were constructed and expressed in E. coli cells. And the biologic assay showed that all of truncated mutants, including the30aa-shorten fragment from N or C terminal of Cry55Aa protein, lose their growth-inhibiting activity against C. elegans. The result suggested that, different to most of other crystal proteins, the nemoticidal crystal protein Cry55Aa is short of an apparent nontoxic region. Based on the result, we suggest that the full-length of Cry55Aa sequence is needed for pest control in genetically modified plant breeding.Secondly, considering that there are many relative proteins to crystal protein Cry5Ba in NCBI protein database, the toxic region of Cry5Ba was predicted by bioinformatics analysis and validated by biological assay. Based on multiple sequence alignment of Cry5Ba and its relative protein in PDB database of NCBI and further three-dimensional structure simulation, the putative toxic region from70aa to700aa was obtained. According to the prediction, a series of truncated fragments of Cry5Ba were constructed and expressed in E. coli and B. thuringiensis cells. The biologic assay result showed that the21aa-shorten fragment from N terminal of Cry5Ba obviously reduced its growth-inhibiting activity against C. elegans, while the10aa-shorten fragment from the same terminal of the Cry5Ba protein exhibited its comparable toxicity to Cry5Ba. These results suggested that, unlike most of other crystal proteins, the partial N terminal sequence of Cry5Ba was necessary for its nemoticidal activity.Thirdly, to reply to the resistance development of pest to crystal proteins from B. thuringiensis, the great efforts of toxicity improvement of Cry5Ba were made in the present thesis. Based on bioinformatics analysis and spacial structure simulation, nineteen amino acid residues in putative domain Ⅱ and Ⅲ of Cry5Ba were mutated to alanine, considering their potential involvement of nemoticidal activity. The biological assay showed that the mutant Y393A and N586A exhibited increased toxicity against C. elegans by3.72and5.58times respectively of that of Cry5Ba. Further study on the mutant N586A demonstrated that the mutant crystal produced by B. thuringiensis improved its solubility in acid pH value when compared to that of wild-type Cry5Ba. In addition, the mutant protein N586A also damaged the host intestine more efficiently than native Cry5Ba. Coupled with confirmation of acid pH value in C. elegans intestinal tract, our results support the presumption that the improved crystal solubility of the mutant N586A might facilitate its dissolution in host acid intestine and then contribute to its increased nemoticidal activity.Finally, as a partial work in my master period, the investigation and evaluation of the biological control of plant root-knot nematode by use of Pasteuria penetrans were included in the present thesis. Our research data showed that most (72.08%) of the root-knot nematodes sampled in Wuhan city were resistant to the P. penetrans endospores imported from Reading University in England, while only25.53%of the plant patristic nematodes were very susceptible to the imported endospores. These results suggested that the P. penetrans display great host specificity which is good for nontarget beneficial animals but bad for effective control of more species of plant root-knot nemotodes. In addition, our further research suggested that the P. penetrans endospores exhibit low selectivity for adhesion position in sensitive host body surface.