الفهرس 
CHAPTER ONEOnly 14 pages are availabe for public view
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Abstract
acillus thuringiensis (Bt), a Gram-positive soil bacterium, can produce
insecticidal crystal proteins called Cry proteins or d-endotoxins during
sporulation stage, which have been extensively used in biological control and a novel
family of insecticidal proteins named vegetative insecticidal proteins (Vip) during its
vegetative stage. This type of protein includes Vip1, Vip2, and Vip3. Vip1 and Vip2 are
binary toxins that have coleopteran specificity, whereas Vip3 toxins have lepidopteran
specificity.
The objectives of this study were to isolate vip1, vip2 and vip3 gene , use Bioinformatics
tools to enhance information about the isolated genes and applying of In-vitro
experiments to examine vip3 gene expression.
The major findings of this study were as follows:
1. A total of 25 soil samples and dead insects were collected from different
geographical locations, isolated and selected look like Bt colonies.
2. A (1.3k) Kb 16S rRNA PCR fragment was amplified using the primer
pair 16srRNA F and R. Amplified fragment was sequenced and total 1.3
kb (nucleotide) sequence was determined. BLAST program was used for
the analysis of the sequence and the homology search showed that it is
like Bacillus thuringiensis serovar entomocidus strain IEBC-T06 00116S
ribosomal RNA gene, partial sequence.
3. The presence of the VIP gene (s) by PCR was done for all 25 isolate
only four gave the product of three VIPs.
4. The full length of vip1 (2.3 Kbp) was isolated and cloned in pGEM®-T
Easy vector. Clones that contain the desired inserts were subjected to
sequencing using the universal primers. Then the vip1 gene was fully
sequenced by the primer walking method using the specific primers. The
similarly of that gene with other vip1 genes was determined by
alignment of the obtained sequence with sequences in the GenBank
database using BLASTn. An alignment of BtVip1 (our isolate) peptide
sequence with other Vip1toxins revealed 75% - 62% identity, except
vip1A (BR) was 100% identity. The phylogenetic tree generated by
Clustal W showed that Vip1A (BR) and BtVip1 (our isolate) were the
same branch as vip1Ba1, vip1Ba2 and vip1Bb. The isolated sequence of
vip3Aa gene was submitted into GenBank and gets an accession
number JN035904.