Characterization Of Crystal Formation Of Bacillus Thuringiensis Crystal Protein Cry51Aa1 And Cloning Of A Novel Crystal Protein Gene Cry65Aa1

Bacillus thuringiensis has unique capability of high expression of Cry protein on various regulated mechanisms, such as crystal formation. And because of the high production of Cry protein, Bacillus thuringiensis has high application value of controlling pest. In this article, we have studied two respects of the work, as follows:1. Effect of the N- and C-terminal amino acid residues on parasporal crystal formation of Bt crystal protein Cry51Aa1.Previous work showed that Bt crystal protein Cry51Aa1, which cloned from Bt strain F14-1, can form bipyramidal crystals without other help proteins. And there are significant differences in molecular weight and the composition of amino acid residues between Cry51Aa1 and Cry1,3,5. In order to find the relationship between amino acid residues of Cry51Aa1 and its crystal formation, we used truncation and alanine scanning mutagenesis analysis to construct a series of mutants, and found that it’s a new mode for bipyramidal crystal formation. The following are the major results:1.1 Cry51Aa1 can not form bipyramidal crystals when truncated the 10 amino acid residues of its C-, N-terminal, respectively;1.2 Cry51Aa1 can form bipyramidal crystals when truncated the 5 amino acid residues of its C-terminal; Cry51Aa1 can not form bipyramidal crystals when truncated the 5 amino acid residues of its N- terminal;1.3 we used alanine scanning mutagenesis analysis to replace the amino acid residues located on 2-7 sites of Cry51Aa1 with alanine, the mutant 2I3L4D,5L6K7S can form irregular inclusion, and the expression level of it has a remarkable declined. It was revealed that the lar amino acid residues located on 4,6 sites of Cry51Aa1, which can form intermolecular salt bond, can help the protein accumulation and are participate in the bipyramidal crystals formation and expression.1.4 we used alanine scanning mutagenesis analysis to replace the amino acid residues located on 8-10 and 297-299 sites of Cry51Aa1 with alanine, the mutant 8L9V10L and 297A298P299T can form irregular inclusion, and the expression level of it was invariant. It was revealed that the aliphatic amino acid residues located on 2,3,5,8, 9,10 sites of Cry51Aa1, which can influence the folding of the protein, are participate in the bipyramidal crystals formation, and not influence the protein expression level. 2. Cloning and expression of a novel Bacillus thuringiensis crystal gene cry65Aa1 from strain SBt003.Sbt003 is a Bt strain stored in our lab, based on bioinformatics analysis of the genome sequence of it, one novel cry gene cry65Aa1, which has the structure of parasporin, was found and it was cloned by PCR. Then cry65Aa1was expressed in the acrystalliferous mutant strain BMB171 and Cry65Aa1 protein was purified for activity analysis. What’s more, we carried out a pilot study of the effect of ORF1 on the parasporal crystal formation of Bt crystal protein Cry65Aa1. The following are the major results:2.1 A novel Bacillus thuringiensis crystal Gene cry65Aa1 was cloned form SBt003, and there is a gene orf1 located downstream. Gene cry65Aa1 encode a polypeptide of 1060 amino acid residues with predicted molecular weights of 117829.60; gene orf1 encode a polypeptide of 512 amino acid residues with predicted molecular weights of 58282.49 Cry65Aa1 showed 28% identity with Cry41Aa1, which is a parasporin exhibit cytocidal activity on cancer cell, and has a structure of the three-domain type, whose amino acid sequence contains five conserved blocks commonly retained in insecticidal Cry proteins; ORF1 showed 98% identity with Cry21Ba1 C-terminal half, and Cry21Ba1 exhibit nematicidal activity.2.3 cry65Aa1 coexpressed with orf1 (located downstream) in the acrystalliferous mutant strain BMB171, then generated BMB1281. BMB1281 can produce parasporal crystal. Then the crystal proteins were analyzed by SDS-PAGE, the result show that BMB1281 can product two major proteins, Cry65Aa1 and ORF1,117 and 58 kDa in size.2.4 Cry65Aa1 can form parasporal crystal with the help of ORF1.2.5 Cry65Aa1 shows low insect growth-inhibitory activity to Helicoverpa armigera.