انتاج بروتين cry1I من بكتريا Bacillus thuringiensis بواسطة التنسل الجيني == Production of cry1I Protein from Bacillus thuringiensis by Gene Cloning
Author name:
نسمة طالب وناس علي
Supervisor name:
محمد الحجاج
General topic:
Biology
Specific topic:
Microbiology - Genetics
Degree:
Master
University:
University Of Basrah - College Of Science
Language:
Arabic
University location:
Basrah
First pages:
24T3576 - p.pdf
Abstract:
Bacillus thuringiensis (commonly known Bt) is a ubiquitous, gram - positive and spore - forming bacterium. The organism produces intracellular crystal proteins, which are toxic to insects, during the stationary phase of its growth cycle. Because of its insecticidal activity, B. thuringiensis has been used as a biopesticide. However, it is still necessary to search for more toxins to control insect orders, which have the ability to develop resistance against such pesticides, and also to provide alternatives for chemical insecticides. The purpose of this study is to isolate B. thuringiensis strains that are collected from Basrah, and to identify the cry genes content of these isolations.In this study, 344 Bacillus species were isolated from 22 soil samples collected from different area from Basrah, fifty six of them (16,2%) were identified as B. thuringiensis strains based on colony morphology , microscopic observation of spore position in the cell and genetic analysis . Most isolations were examined by multiplex PCR using for cry 1, cry 2, and cry 9 universal primers in order to identify the type of cry gene content of these isolations. 82% of the isolations amplified cry1 gene, 76% amplified cry9 and 21% amplified cry2 genes.In this study cloned cry1I gene is using specific primer to amplify full length of gene. The cry 1I gene (2169 bp) amplified product was inserted in to the Pst1 and BamH1 sites of pdrive cloning vector joining technique to produce the recombinant vector. The cloning vector then transformed in to E.coli HB101 and the transformant cells colonies were selected by ampicillin sensitive phenotype, the efficiency of transformation was also determined to be 7,8 ×105cfu/μg. After that the cry1I protein is purified from LB broth media supported with ampicillinand used this protein against Tuta absoluta larva within concentrations (100 μg/ml, 150μg/ml, 200μg/ml) .The effectiveness of the toxin is to kill the larvae were in the concentration of (200μg/ml) in the 24 hours after treatment, while least toxicity in the concentration (100μg/ml) need three days to kill all larva.
