Classification Of CTC Strain And Construction For The Expression Vectors Of Its S-layer Protein Genes

This study involves two parts, one is that we finished the identification of CTC strain by comparing the sequence of the spacer region between 16S rRNA and 23S RNA, the other is that we combined ctcl gene and ctc2 gene to the shuttle vector pHT304 for functional analysis, or ctcl and ctc2 separately.The CTC strain isolated by our lab is identified as Bacillus thuringiensis subsp. finitimus (serotype H2) by the Pasteur Institute. After our further study, we found that although the CTC strain had the ability to form parasporal crystal, it was unstable. This crystal protein is not encoded by the plasmid-carried cry genes but by the genes encoding S-layer protein, and no toxicity to insects was detected. All these treats are quite different from others in Bacillus thuringiensis. So we doubt the conclusion that CTC strain belongs to Bacillus thuringiensis. In order to show the most appropriate identification status, we analyzed the spacer sequence of 16S-23S rRNA-the new hotspot technology in the international phylogenetic field. Some combinations were made, and seven candidate strains from these combinations could be amplified by PCR through the special primers we designed. And the productions of PCR were cloned into vector for sequencing. This strategy not only allowed us to clear the differences between the seven strains' sequences, but also to carry out clustering analysis by quering the data base for heterogenous sequences. The results display that the 16S rRNA and 16S rRNA-23S rRNA sequences of CTC strain and T02 strains are heterogenous, where discrepancy exists between the conclusion from our study and of the Pasteur Institute's . Additionally, there is something interesting that CTC strain did not completely unclassified from others of Bacillus thuringiensis, but between Bacillus thurgiensis and Bacillus cereus. And in terms of the fact that the crystal toxicity of T02 to the insect has not been detected yet, we conclude that CTC strain is belongs to Bacillus cereus, and mostly trends to a transitional type between Bacillus cereus and Bacillus thuringiensis which is of great value to the taxonomy.S-layer which universally exits in prokaryote has a membrane subunit structure, locates on the cell surface and in crystal lattice arrangement assembled by the same subunits. Its planar crystal unit is assembled by a single glycoprotein. Because of the highregularity of the structure, S-layer presents the simplest model of the membrane formation evolutionally. Nevertheless, it was reported that the S-layer lose easily when bacteria were cultivated in lab, which would not happen when growing under the nature competitive environment. Though generally considering that S-layer is located on the cell surface, the strain separated in our lab is not so. The S-layer protein is not only located on the cell surface but also formed crystal in the cell. We've already cloned two S-layer genes and these two genes have high homology with the two genes finding in Bacillus anthracis. But the S-layer in this Bacillus anthracis is under normal configuration. Why does the S-layer protein in this strain form crystal? And this crystal is composed of the single S-layer protein or of the two S-layer proteins? And the form of this crystal is the result of over secret or because it could not locate on the surface so it went into the cell? In order to answer these questions we designed some vectors including: only ctcl gene contained, only ctc2 gene contained, and both ctcl gene and ctc2 gene contained. All these work facilitate the further research of this field.