Oral presentation
27
GENETIC ANALYSIS OF THE CRY GENES OF THE
ENTOMOPATHOGENIC BACILLUS THURINGIENSIS AGAINST
Helicoverpa armigera
I.M. Khalilov, F.B. Kobilov, N.S. Akhmedova
Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan, 7b
Abdulla Qadiri Street, Tashkent 100128
Microorganisms are currently being used to produce ecologically green agricultural products.
Among such microorganisms,
Bacillus thuringiensis
(Bt) bacterium is the best biological agent
against pest insects.
For this reason, it is possible to achieve many desired results by
comprehensively studying this bacterium's morphological and genetic characteristics. That is why
this bacterium has been the focus of the attention of many scientists.
Bacillus thuringiensis
is a gram-positive, rod-shaped, spore-forming bacterium found
worldwide in soil, water, dead insects, hay,
tree leaves, various conifers, and diverse
ecosystems, including insectivorous mammals, as well as humans with severe necrosis.
It has been found that it occurs and develops in the tissues and as a probiotic in the
composition of cow's milk, in environments contaminated
with phenolic compounds
and various pesticides. An entomopathogenic feature of Bts is that they produce
crystalline proteins (Cry). These proteins cause the death of harmful insects. Cry genes
code these Cry proteins.
Using the modified Marmur method, genomic DNA was isolated from
Bt
strains,
specific primers for cry1Aa-I, cry1Ab, cry1Ac-I, cry9U-I, and cry2
genes were
designed and PCR amplification was performed. When the partial sequence of the
cry1Ab gene of strain
Bt94
was compared in the NCBI database, 93.53% similarity was
observed with the genes numbered AY007686.1, M97880.1, and MT226609.1, and
93.41% with the gene numbered EU679502.1. The cry1Ab gene was compared using
Slustal Omega online software to find SNP variation.
According to the results of the analysis, it was found that the
Bt94
cry1Ab gene was
mutated at 32 points from the nucleotide sequence of other compared genes. In this
case, at nucleotide 83 (T/C), at 87 (A/S), at 154 (T/S), at 195 (G/A), at 227 (T/G), at 282 (
G/A), 315 (G/S), 318 (T/S), 357 (A/S), 361 (A/S), 372 (S/T), 381 (A/G), 388 (G/A), 435
(S/T), 440 (T/A), 461 (A/G), 493 (A /G), 501 (T/S), 517 (T/G), 552 (S/T), 555 (T/S), 564
(S/T), 567 (T/G), 571 (G/A), 576 (A/G), 591 (G/A), 597 (S/A), 621 (T/ A), it was observed
that there was a mutation at 674 (G/T), 682 (S/G) and 685 (A/G) nucleotides, and only
the
Bt94
cry1Ab gene changed from the others at these nucleotide positions with 20
transitions and 12 transversions. the mutation was analyzed.
It should be noted that the nucleotide sequence of the cry1Ab gene of the
Bt94
strain,
assuming that the primers encoding the cry1Ab gene also encode the cry1s gene, it can
be seen in the NCBI database that the gene product identified as the cry1Ab gene
belongs to the variant of the cry1s gene.
Thus, when the partial genes
Bt1
-cry1Ac,
Bt18fo
-cry1Ab,
Bt26
-cry1Aa,
Bt31
-cry2,
Bt84
-
cry1Ac,
Bt91
-cry9U,
Bt93
-cry9U, and
Bt94
-cry1Ab
were constructed, a phylogenetic tree
was created using the Mega-4 statistical program. All identified genes were observed to
match their corresponding cry gene classification. In addition, it
was found that all studied
genes are in 1 cluster, respectively cry genes. The identified partial cry genes are a good
scientific resource for future whole-genome sequencing and expression studies.