Organizing co mmittee

CLONING ANALYSIS OF CRY1AA GENE OF 
Bacillus thuringiensis 1FO STRAIN 
 
F.B. Kobilov, I.M. Khalilov, M.M. Nazirov, R.A. Alamuratov, N.Sh. Azimova 
 
Institute of Microbiology of the Academy of Science of the Republic of Uzbekistan. 7b 
Abdulla Qadiri Street, Tashkent 100128 
It is known that biological agents, in particular, microorganisms, are used in the care 
of agricultural crops, fruit and ornamental trees, especially in protecting them from 
various pests. 
Bacillus thuringiensis
bacterium also contains genes encoding many anti-
pest proteins, and screening and research on the genetic characteristics of 
B. 
thuringiensis
have been carried out for more than 30 years. 
B. thuringiensis
(Bt) is an aerobic, spore-forming, gram-positive, and entomopathogenic 
bacterium that produces parasporal crystal proteins or δ-endotoxins (Cry). 
Based on amino acid sequence similarity, 79 cry gene families (cry1-cry79) with 
more than 770 cry genes have been characterized to date. These toxins exhibit strong 
toxicity against Lepidoptera, Diptera, Coleoptera, Hymenoptera, Orthoptera, Hemiptera, 
and Nematodes, which are considered to be various insect pests. The Cry1Aa gene 
encodes the CRY1Aa protein, which is toxic to pests of the Lepidoptera group. 
Firstly, genomic DNA was isolated from 
B. thuringiensis
1fo
strain and specific 
primers were designed for screening Cry1Aa gene and PCR amplification was 
performed. Cry1Aa fragment was ligated into the vector (4634 bp) and chemically 
transformed into 
E. coli DH5a
strain. Cry1Aa gene ligation was detected by oncolony 
PCR from colonies grown on LB agar medium with selective kanamycin (Fig. 1 A). 
The resulting colonies were planted in LB (kanamycin) liquid medium. Then, plasmid 
DNA was isolated and restricted using 
KpNI
- 
BamHI
enzymes (Fig. 1 B). 
Figure 1. A-Cry1Aa gene oncolony PCR product, B- Cry1Aa gene restriction analysis: 
1-
KpNI
and 
BamHI
, 2-
BamHI
, 3-uncut plasmid 
Figure 1 shows that the Cry1Aa gene was efficiently cloned, and the PCR and 
restriction enzyme products were of the same size as expected for the Cry1Aa gene. 
According to the literature that CRY1Aa protein has an active effect on other pests 
besides 
Helicoverpa armigera
and 
Helicoverpa zea
. For example: 
Plutella xylostella
insects fed on cabbage and brussels sprouts transformed with Cry1Aa genes were 
reported to die 100% within 72 hours. 
In conclusion, the cloning of the Cry1Aa gene of 
Bacillus thuringiensis
is an 
important scientific achievement in the field of insect pest control and can be a powerful 
tool for sustainable agriculture. 
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