Научная статья на тему 'IDENTIFICATION AND POPULATION VARIABILITY OF LOCAL BACILLUS THURINGIENSIS STRAINS'

IDENTIFICATION AND POPULATION VARIABILITY OF LOCAL BACILLUS THURINGIENSIS STRAINS Текст научной статьи по специальности «Биологические науки»

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Ключевые слова
Bacillus thuringiensis / entomopathogen / 16S rRNA gene / taxonomy / identification. / bacillus thuringiensis / энтомопатоген / ген 16S рРНК / таксономия / идентификация.

Аннотация научной статьи по биологическим наукам, автор научной работы — Khalilov Ilkhom Mamatkulovich, Qobilov Fazliddin Bozorovich, Azimova Nodira Shoims Girl, Nazirov Muhammad-Latif Marufs Son, Mardonov Ikrom Hasans Son

this research involved the use of Bacillus thuringiensis (Bt) strains Bt1, Bt18fo, Bt26, Bt31, Bt84, Bt91, Bt93 and Bt94 that were isolated from different regions of Uzbekistan. To identify the eight isolated Bt entomopathogenic strains, the 16S rRNA gene 16S-23S internal transcribed spacer (ITS) region nucleotide sequences were partially sequenced, and a phylogenetic tree was constructed. It was found that there were differences partial nucleotides in the 16S rRNA gene of eight Bt strains. However, they are 99.51-100% similar to bacteria belonging to the Bacillus cereus group. When the Bt strains were aligned with each other in the Clustal Omega online program, differences in the 16S rRNA gene were noted even though they were isolated from the same ecological zone. In addition, PCR analysis of cry1Aa, cry1Ab, cry2B, cry9Ba-I and cry1Ac genes showed the presence of cry1Ab and cry9Ba-I genes in strains Bt1, Bt18fo, Bt26, Bt31, Bt91, Bt93 and Bt94. The cry2B gene was detected only in the Bt31 strain, while the cry1Ac gene was detected in the Bt1 and Bt84 strains.

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ИДЕНТИФИКАЦИЯ И ПОПУЛЯЦИОННАЯ ИЗМЕНЧИВОСТЬ МЕСТНЫХ ШТАММОВ BACILLUS THURINGIENSIS

в данном исследовании использовались штаммы Bacillus thuringiensis (Bt) Bt1, Bt18fo, Bt26, Bt31, Bt84, Bt91, Bt93 и Bt94, выделенные из разных регионов Узбекистана. Для идентификации восьми выделенных энтомопатогенных штаммов Bt были частично секвенированы нуклеотидные последовательности области 16S-23S (ITS) гена 16S рРНК 16S и построено филогенетическое дерево. Было обнаружено, что в гене 16S рРНК восьми штаммов Bt имеются различия частичных нуклеотидов. Однако они на 99,51-100% сходны с бактериями, относящимися к группе Bacillus cereus. Когда штаммы Bt были сопоставлены друг с другом в онлайн-программе Clustal Omega, были отмечены различия в гене 16S рРНК, даже если они были изолированы из одной экологической зоны. Кроме того, ПЦР-анализ генов cry1Aa, cry1Ab, cry2B, cry9Ba-I и cry1Ac показал наличие генов cry1Ab и cry9Ba-I у штаммов Bt1, Bt18fo, Bt26, Bt31, Bt91, Bt93 и Bt94. Ген cry2B обнаружен только у штамма Bt31, тогда как ген cry1Ac обнаружен у штаммов Bt1 и Bt84.

Текст научной работы на тему «IDENTIFICATION AND POPULATION VARIABILITY OF LOCAL BACILLUS THURINGIENSIS STRAINS»

IDENTIFICATION AND POPULATION VARIABILITY OF LOCAL BACILLUS

THURINGIENSIS STRAINS Khalilov I.M.1, Qobilov F, B.2, Azimova N. Sh. 3, Nazirov M. M. 4, Mardonov I.H. 5,

Akhmedova N.S.6, Turaeva S.Sh.7.

1Khalilov Ilkhom Mamatkulovich - PhD in Biology, Senior Researcher; 2Qobilov Fazliddin Bozorovich - PhD student; 3Azimova Nodira Shoim's girl - PhD in Biology, Senior Researcher; 4Nazirov Muhammad-Latif Marufs son - PhD student; 5Mardonov Ikrom Hasan's son - Junior researcher, MOLECULAR BIOLOGY DEPARTMENT, INSTITUTE MICROBIOLOGY OF ACADEMY SCIENCES OF UZBEKISTAN, TASHKENT, REPUBLIC OF UZBEKISTAN 6Akhmedova Navbakhor Soliyboy's girl - undergraduate, NATIONAL UNIVERSITY OF UZBEKISTAN; 7Turaeva Sanobar Sharif's girl - undergraduate, KARSHI STATE UNIVERSITY, UZBEKISTAN, KARSHI, REPUBLIC OF UZBEKISTAN

Abstract: this research involved the use of Bacillus thuringiensis (Bt) strains Bt1, Bt18fo, Bt26, Bt31, Bt84, Bt91, Bt93 and Bt94 that were isolated from different regions of Uzbekistan. To identify the eight isolated Bt entomopathogenic strains, the 16S rRNA gene 16S-23S internal transcribed spacer (ITS) region nucleotide sequences were partially sequenced, and a phylogenetic tree was constructed. It was found that there were differences partial nucleotides in the 16S rRNA gene of eight Bt strains. However, they are 99.51-100% similar to bacteria belonging to the Bacillus cereus group. When the Bt strains were aligned with each other in the Clustal Omega online program, differences in the 16S rRNA gene were noted even though they were isolated from the same ecological zone. In addition, PCR analysis of cry1Aa, cry1Ab, cry2B, cry9Ba-I and cry1Ac genes showed the presence of cry1Ab and cry9Ba-I genes in strains Bt1, Bt18fo, Bt26, Bt31, Bt91, Bt93 and Bt94. The cry2B gene was detected only in the Bt31 strain, while the cry1Ac gene was detected in the Bt1 and Bt84 strains. Keywords: Bacillus thuringiensis, entomopathogen, 16S rRNA gene, taxonomy, identification.

ИДЕНТИФИКАЦИЯ И ПОПУЛЯЦИОННАЯ ИЗМЕНЧИВОСТЬ МЕСТНЫХ

ШТАММОВ BACILLUS THURINGIENSIS Халилов И.М.1, Кобилов Ф.Б.2, Азимова Н.Ш.3, Назиров М.М.4, Мардонов И.Х.5,

Ахмедова Н.С.6, Тураева С.Ш.7.

:Халилов Ильхом Маматкулович - кандидат биологических наук, старший научный сотрудник;

2Кобилов Фазлиддин Бозорович - аспирант; 3Азимова Нодира дочь Шоима - кандидат биологических наук, старший научный сотрудник; 4Назиров Мухаммад-Латиф сын Маруфа - аспирант; 5Мардонов Икром сын Хасана - младший научный сотрудник, Лаборатория молекулярной биологии, институт микробиологии академии наук Узбекистана,

г. Ташкент, Республика Узбекистан 6Ахмедова Навбахор дочь Солыбоя - магистрант, Национальный университет Узбекистана; 7Тураева Санобар дочь Шарифа - магистрант, Каршинский государственный университет, г. Карши, Республика Узбекистан

Аннотация: в данном исследовании использовались штаммы Bacillus thuringiensis (Bt) Bt1, Bt18fo, Bt26, Bt31, Bt84, Bt91, Bt93 и Bt94, выделенные из разных регионов Узбекистана. Для идентификации восьми выделенных энтомопатогенных штаммов Bt были частично секвенированы нуклеотидные последовательности области 16S-23S (ITS) гена 16S рРНК 16S и построено филогенетическое дерево. Было обнаружено, что в гене 16SрРНК восьми штаммов Bt имеются различия частичных нуклеотидов. Однако они на 99,51-100% сходны с бактериями, относящимися к группе Bacillus cereus. Когда штаммы Bt были сопоставлены друг с другом в онлайн-программе Clustal Omega, были отмечены различия в гене 16S рРНК, даже если они были изолированы из одной экологической зоны. Кроме того, ПЦР-анализ генов cry1Aa, cry1Ab, cry2B, cry9Ba-I и cry1Ac показал наличие генов cry1Ab и cry9Ba-I у штаммов Bt1, Bt18fo, Bt26, Bt31, Bt91, Bt93 и Bt94. Ген cry2B обнаружен только у штамма Bt31, тогда как ген cry1Ac обнаружен у штаммов Bt1 и Bt84.

Ключевые слова: bacillus thuringiensis, энтомопатоген, ген 16SрРНК, таксономия, идентификация.

INTRODUCTION

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming, rod-shaped soil bacterium that is found in various ecological systems throughout the earth, including in soil, water, dead insects, the leaves of deciduous trees, the internal tissues of some plants (endophytes) and milk. [1-4]. Bt bacteria produce broad-spectrum insecticidal proteins that are active against the larvae of a wide variety of insects and have sometimes been observed to affect insects other than these as well. The products made on the basis of these bacteria have become the most frequently sold biological insecticides worldwide [4,5], as they include genes that encode proteins with insecticidal properties and introduce the possibility of creating new types of transgenic plants that are resistant to pests as a result of their transfer to plants [6]. Based on phenotypic and genotypic analyses, Bt species belong to the Bacilus cereus group of bacteria. However, the identification of vegetative and spore-forming Bt bacterial strains with B. cereus species through morphological [7], phenotypic [8] or genotypic methods is very difficult [9]. Bt strains can be identified mainly by the synthesis of cry (cyt or vip)-proteins in crystal form [10]. Comparisons of the Bt strains belonging to the B. cereus group according to 16S rRNA and gyrB gene nucleotide sequences based on arbitrarily primed (AP)-PCR, including box element (BOX) and enterobacterial repetitive intergenic consensus (ERIC), and saAFLP methods showed that Bt strains were divided into separate groups. Indeed, these methods have been found to be suitable for detecting differences in Bt bacteria within species [11]. Differentiation of Bt bacteria within species was also reported using repetitive element (REP)-PCR and ERIC-PCR, but no correlation was observed between the isolation sites and insect toxicity [12]. Since Lepidoptera, Diptera, Coleoptera, Hemiptera, Hymenoptera and similar pests cause great damage to a number of plants worldwide, introducing genes that encode insecticidal proteins into plants is a very effective way to protect plants from these pests [13, 14, 15]. Because of the presence of Bt isolates with unique proteins of insecticidal genes in every ecological system, research groups around the world are constantly studying different biological samples to find such new Bt isolates [16, 17, 18].

The purpose of this research work is to identify the 16S and 23S (ITS) region of the rRNA partial gene of the Bt1, Bt18fo, Bt26, Bt31, Bt84, Bt91, Bt93 and Bt94 strains that was isolated from different areas of Uzbekistan through molecular genetic methods. It is also necessary to identify some cry genes of these strains and to determine the difference between these strains and the B. cereus and other Bacillus species.

MATERIALS AND METHODS

Strains of Bacteria and Medium

The Bt1, Bt18fo, Bt26, Bt31, Bt84, Bt91, Bt93 and Bt94 strains were examined as the object of the study. The Bt1, Bt18fo, Bt26 and Bt31 strains were isolated from the fields of honey waxworm-breeding enterprises around Kibrai, in the Tashkent region. The Bt1 strain was isolated from the insect Helicoverpa armigera, and the remaining strains, Bt18fo, Bt26 and Bt31, were isolated from the dead insect Galleria mellonella. The Bt84, Bt91, Bt93 and Bt94 strains were isolated from Lymantria dispar in the forest zone of Ugam-Chotkal National Park in the Tashkent region. Bacteria were grown in a peptone nutrient medium (PB): Peptone-1.0%, glucose-0.6%, NaCl-0.5%, K2HP04-0.05 % and MgSO4-0.02% (pH-7.0).

DNA isolation and PCR amplification of the 16S rRNA gene of B. thuringiensis strains

DNA extraction of eight Bt isolates was performed according to the Wizard Genomic DNA Purification Kit [19]. Extracted DNA samples were stored at -20 °C. The following primers were used in the PCR reactions: forward 5'-TCTCAGTCGGATTGTAGGC-3' and reverse 5'-ATCGACTCCTAGTGTCAAGG-3' [20]. Amplification reactions were conducted in a Mastercycler thermal cycler (Bio-rad, USA). The PCR products were then detected on a 1.5 % agarose-EtBr gel, and the amplified PCR products were observed in a GelDoc system (Bio-rad, USA). PCR amplification was carried out as described previously [20].

Table 1. PCR-amplification of cry genes of Bt strains was carried out using the following primers.

Gen name Sequence (5' ') bp Reference

cry1Aa-I (F) TGCATAGAGGCTTTAAT 1500 [20]

cry1Aa-cI (R) CAGGATTCCATTCAAGG

cry1Ab-I (F) TCGGAAAATGTGCCCAT 858

cry1Ab-I (R) AATTGCTTTCATAGGCT

cry9Ba-I (F) CGGTGTTACTATTAGCGAGGGCGG 351 [21]

cry9Ba-I (R) GTTTGAGCCGCTTCACAGCAATCC

cry2-I (F) GGATCCATATGAATAGTGTATTGAAT 1926

cry2-I (R) GGATCCATATGAATAGTGTATTGAAT

The PCR products were then electrophoresed on a 1.5% agarose-ethidium bromode (EtBr) gel and the amplified PCR products were detected in a GelDoc system (Bio-rad, USA). Sequencing was carried out using the BigDye Terminator v.3.1 cycle sequencing kit and Applied Biosystems® Genetic Analyzers, 3130 series sequencer (Thermo Fischer Scientific, USA).

RESULTS

In recent years, a large number of databases have been developed for 16S rRNA, and based on these the phylogenetic position of bacteria is being identified. Because most 16S rRNA genes make up about 1,500 base pairs, some branches of the genes as domains (Archaea, Eucaea and Bacteria) reveal broad characteristics of phylogenetic groups, while other branches of the gene reveal narrower characteristics of groups, such as phylum, class and genus. A 16S rRNA gene sequence similarity of 97% provides information about the genus of the bacteria, while a similarity above 99% provides information about the species [22].

PCR products of eight B.thuringiensis strains

To identify the 16S and 23S (ITS) region of the rRNA partial gene of the Bt bacterial strains, genomic DNA was isolated and a PCR product over 400 bp was obtained using primers (Figure 1).

lkb 1 2 3 4 5 6 7 8

Fig. 1. The 1kb marker. 1-Bt1, 2-Bt18fo, 3-Bt26, 4-Bt31, 5-Bt84, 6-Bt91, 7-Bt93 and 8-Bt94 and the PCR product of

16S-23S (ITS) part of rRNA gene.

To compare the phylogenetic position of Bt bacterial strains to each other and to other species of bacteria, the 16S-23S (ITS) part of the rRNA gene was sequenced, and a 410 bp nucleotide sequence was determined. The results of the nucleotide sequences of the 16S rRNA gene 16S-23S (ITS) that were obtained were placed in the National Center for Biotechnology Information (NCBI) database, and the following accession numbers were obtained: Bt1 (MN244691), Bt18fo (MN244692), Bt26 (MN244693), Bt31 (MN244694), Bt84 (MN244695), Bt91 (MN244696), Bt93 (MN244697) and Bt94 (MN244698).

Multiple sequence alignment of eight B.thuringiensis strains

The 16S rRNA gene that was isolated from the Bt strains studied experimentally was aligned in the Clustal Omega online software program for comparison based on the partial sequence of the strains (Figure 2).

CLUSTAl. 0(1.2.4) multiple sequence alignment

Bt91 165 TCTCAGTTCGGATrGTAGGCTGCAACTCOCCTACATGAAGCTGGAATCGCTAGTAATCGC «0

Bt94 ttS TCTCA£TTCGGATTGTAGGCTGCAACTCGCCTACAT<ïAAGCTGiïAATCGCTA£;TAATCGC CO

Bt31_16S TCTCAGTTCGGATTGTAGGCTG^^ACTCGCC^ACATGAAGCTGGAATCGCTAGTAATCGC 60

BU16S TCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGC 60

Btl 8iO J6S TCTCA^TTCWATTGTAGGCTGCAACTOOCCTACATGAAGCTGGAATCGCTJVSTAATCGC 60

Bt26 I6S TCTCAGTTCGGATTGTAGGCTGCAACTOGCCTACATGAAQCTGGAATCGCTAûTAATCGC 60

Bt93 I6S TCTCAGTTCGGATrGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGC 60

Bt 84 1 65 TCTCAGTTCGGATTGTAGGCTGCAACTCOCCTACATGAAQCTGCAATCGCTAfiTAATCGC 60

Bt.91_16S G5ATCAGCATWXGCGGTGAATACGTTCCCGQGCCTTGTACACACCGCCCGTCACACCAC Î20

Bt94_16S CU^TCAIKATGCCiKGCTGAATACOTTCCCGOGCCTTGTACACACCGCCCGTCACACCAC '.20

Bt31 16S G3ATCAGCATGCCGCGGTGAATACGTTCC"GOGCCTTGTACACACCGCCCGTCACACCAC 120

B11Ï6S GSATCAGCATtKCGCGGTGAATACGTTCCCGQGCCTTGTACACACCGCCCGTCACACCAC 120

BtlSioliS GSATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAC 120

Bt26_16S GGATCAGCA7GCCGCGCTGAATACGTTCCCGOGCCTTGTACACACCGCCCCTCACACCAC 120

Bt93_165 GGATCAGCATGCCGCGGTGAATACGTTOCCGQGCCTTGTACACACCGCCCGTCACACCAC 120

Bt84 16S G3ATCAGCA7GCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAC 120

Bt91 16S ■194 165 Bl31~16S Btl_16S Btl8fo_16S Bt26_l65 Bt93_16S Bt84 165

GAGAOrtTCTAACACCCGAAaTCGCTGOGGTAACCT T? I tGGAGCCAOCCQCCTAAGGTG 180

GAGAGTTrCTAACACCCGAAGTCGOTGOGGTAACl IT ï ITOGAOCCAGCCGCCTAAOGTG 180

GAGAÎÏTTTGTAACACCCGAAGTCGGTQGGGTAACCTTTTTGGAGCCAÎSCCGCCTAAGGTO 180

GAOACrrWrAACACCCOAAGTCOGrQaGSTAACCT ITT IGOASCCAaCCOCCTAAOOTG 180

GAGAGTTTGTAACACCCGAASTCGGTGGGGTAACCTTTTTGGAjGCCAGCCGCCTAAGGTG 180

GAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTG 180

GAGACTTTGr?AACACCCGAAGTCGGTOQOGTAAC117TTI TGGAGCCAOtCQCCTAAGGTG 180

GAGAG?TTG?AACACCCGAA£TCGCTG<3GGTAACCTT?TTGGAGCCAGCCGCCTAAGGTG 180

BC9I I6S CaACAGAraATTaGGGTGAASTCGTAACAAGCrAGCCCTATCGaAAGCTGCOSCTOGATC 240

Bt94 16S ÛSACAGATGATTaGGGTGAASTCGTAACAAGGTASCCGTATCGGAAGGrGCGGCTGGATC 243

Bt31165 GGACAGATGATTGGGGTGAAGTCG7AACAAGGTAGCCGTATCGGAAGGTGCQGCTGGATC 240

Btl_Ï6S GSACAGATGATTOGGGTGAAGTCGTAACAAGGTAGCCGTATCG&AAGGTGCQGCTQGATC 240

BtlSto liS GSACAGATGATTGGGGTGAAGTCOTAACAAGGTAIjCCCTATCGGAAGGTGCGSCTGGATC 240

Bt26 16S GGACAGATGATTQGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCOSCTGGATC 240

Bt93l6S GGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGliAAGGTGCOSCTOGATC 240

Bt.84 ICS GGACAGAT^TTœGGTGAAGTCCTAACAAGGTASCCGTATCGaAAGCTGCaSCTOGATC 243

Bt91 _î63 ACCTCCTTTCTATGGAGAATTGATQAACOCTGTTCATCAATAA-AOTTTCCGTGTTTCGT 299

Bt94 16S »eCICClîtCTATGGAGAATTaATGAACGetGTTCATCAATAT^AGrrTCCGTfllI'rCGT 300

Bt31 16S ACCTCCTTTCTATGGAGAATTGATGAACGC7GTTCATCAATAA-AGTTTCCGTGTTTCGT 299

Btl_16S ACCTCCTTTCTATGGAGAATTGATGAACGCTGTTCATCAATAA--AG7TTCCCTGTTTCGT 299

BtHfo l&S ACCTCCrrrCTATGGAGAATTQATWA^TGTTCATCAATAA-AGTTTCCCTGrrrCGT 299

Bt26_lCS ACCTCCTTTCTATGGAGAATTGATGAACGCTGTTI^TCAATAA-AGTTTCCGTGTTTCGT 299

Bt93 16S ACCTCCTTTCTATGGAGAATTGATGAAOGCTGT7CATC AAT AA-AGTTTCCGTGTTTCGT 299

Bt84 165 ACCTCCTTTCTATGGAGAATT3A7GAACGC7GT7CATCAATAA-AG7TTCCGTGTTTCGT 299

Bt.91 165 Bt94_16S Bt3i 16s Bll_Î6S Btl8fo_16S Bt26_liS Bt93 16S Bt 84 163

TTTGT7CAGTTTTGAGAGAACTATCTCTCATATAT AAATGTATGTTCTTTGAAAAC7AGA 359

TTTGT7CAG7TTTGAGAGAACTA7CTCTCATATATAAATGTATCTTCTTTGAAAAC7AGA 360

TTTGTTCAGTTTTGAGAGAACTATCTCTCATATATAAATCTATCTTCTTTGAAAACTAGA 359

TTTGTTCAGTTTTGAGAGAACTA7CTCTCATATATAAATGTATCTTCTTTGAAAAC7AGA 359

TTTGTTCAGTTTTGAGAGAACTATCTCTC^ATATAAATGTATGTTCTTTGAAAACTAGA 359

TTTC»TTCA<7rTTTGAGAGAACTATCTCTCATATATAAAT(ÏTATCTTCTTTGAAAACTAGA 359

TTTGrrTCAGTTTTGAGAGAACTATCTCTCATATATAAATGTATtSTTCTTTGAAAACTAGA 359

TTTGTTCAGTTTTGAGAGAACTATCTCTCATATATAAATGTATGTTCTTTGAAAACTAGA 359

Bt91_16S TAACAGTCTAGCTCATATnTlfsfTTTt^JrTTGG-TAAGTTAGAAAGG

Bt94 16S TAACAGTGTAGCTCATATTTTTTAATTTTTAGTTTGGnAAGTTAGAAAGG

Bt 31_165 TAACAGTGTAGCTCATATTTTTTAAT7m AGTTTGQTTAAGTTAGAAAGG

BtllCS TAACAGTGTAGCTCATATnfrTAATTTTTAGTTTOGrrAAGTTAGAAAGG

BtlSfo 16S TAACAGTGTAGCTCATATTTTTTAATTTTTAjGTTTGGTTAAGTTAGAAAGG

Bt26_16S TAACAGTGTAGCTCATAT7TTTTAAH IT IAGTTTGGTT AAGTT AGAAAGG

Bt91_16S TAACAGTGTAGCTCATATTTTTTAATTTTTACTTTGGTTAAGTTAGAAAGG

Bt 84 16S TAACAGTCTAGCTCATATTTTTTAATTTTTAjGT? I go: IAAGTTACAAAGG

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409 411

410 410 410 410 410 410

Fig. 2. Intercomparisons of partially sequenced 16S rRNA gene nucleotide sequences of Bt strains.

However, the nucleotide sequences of the Bt31 strain 23 - (C/T) and 32 - (T/C) underwent transversion mutations and differed from the nucleotide sequences of other strains. Also, the Bt94 strain, unlike other strains, underwent an inversion mutation at the 284th position, creating a new nucleotide (-/A). In strain Bt91, a transversion mutation was detected at nucleotide positions 382 - (T/A), 384 - (A/T) and 389 - (T/A), while a transition mutation was noted at nucleotide position 390 - (A/G). It was also found that the Bt91 strain, unlike other strains, had a deletion at position 391 (G/-), and in the Bt1 strain the nucleotides at position 330 - (T/A) were changed by transversion. No single nucleotide polymorphisms (SNPs) were observed in the nucleotide sequences of strains Bt18fo, Bt26, Bt84 and Bt93.

Phylogenetic Tree

Based on the nucleotides that were identified, it was noted that the Bt strains differed from one another by one or five nucleotides. Based on the nucleotide sequences of the 16S rRNA gene that were obtained, a phylogenetic tree was constructed using the maximum likelihood statistical method with the Mega 4 bioinformatics program (Figure 3).

MG543839 1 Eschenchia coli strain NW A26 16S

Fig. 3. The phylogenetic tree of the local Bt strains using the Mega 4 bioinformatics software maximum likelihood statistical

method

All strains were found to be 99.8% identical to B. thuringiensis and B. cereus when checked against the NCBI database. Also, the Bt1, Bt26, Bt84, Bt93 and Bt94 strains showed 99.78% similarity to B. cereus bacteria, the Bt18fo and Bt91 strains showed 99.77-100% similarity to B. cereus bacteria and the Bt31 strain showed 99.51% similarity to B. cereus bacteria. As seen in Figure 3, the nucleotide sequences of all sequenced strains are very similar, amounting to 99.9%.

The phylogenetic tree showed that the Bt18 strain was observed to form a new clade that was separate from other strains. Bt strains were found to be 99% identical to strains of B. cereus, B.tropicus, B.subtilis, B.albus and B.mobilus. It was noted that Pseudomonas putida and Escherichia coli bacteria, which are considered to be out of the Bacillus group, branch separately in the phylogenetic tree.

Since some local strains of Bt bacteria have differences in the nucleotide sequences of the 16S rRNA gene, the presence of cry1Aa, cry1Ab, cry2B, cry9Ba-I and crylAc genes was analysed using PCR to determine the differences in the content of the cry toxins produced by these strains.

Table 2. PCR products of cry genes of Bt bacteria.

№ Used strains PCR amplified cry genes

cry1Aa-I crylAb cry9Ba-I cry2 Cry1Ac-I

1 Bt 1 - + + - +

2 Bt 18$o - + + - -

3 Bt 26 + + + - -

4 Bt 31 - + - + -

5 Bt 84 - - + - +

6 Bt 91 - + + - -

7 Bt 93 - + + - -

8 Bt 94 - + + - -

(- cry gene is not present; + cry gene is present)

Table 2 shows that in the PCR analysis of crylAa, crylAb, cry2B, cry9Ba-l and crylAc genes, the crylAa gene was found only in the Bt26 strain, although the presence of crylAb and cry9Ba-l was observed in the Btl, Bt18fo, Bt26, Bt3l, Bt9l, Bt93 and Bt94 strains. While cry2B and crylAb genes were found in the Bt3l strain, the crylAc gene was detected in the Btl and Bt84 strains. It should be noted that three cry genes were found in Btl (crylAb, cry9Ba-Iand crylAc) and Bt26 (crylAa, crylAb and cry9Ba-I) strains used in the experiment.

DISCUSSION

Gypsy moth larvae can damage more than 300 species of trees for 10 weeks a year [24]. During such outbreaks, high densities of caterpillars can be a nuisance and a health concern to homeowners, especially those who live in or adjacent to forested areas [25]. To control these insects in natural forest areas, it is important to use biological insecticides instead of chemical pesticides to avoid destroying the ecological systems. One method of biological control in forest zones is through the use of preparations made with B.thuringiensis bacteria. Therefore, genetic identification of these bacterial species is important along with studying cry genes that express insecticidal activity. However, difficulty with regard to genetic identification of bacteria that belong to the B. cereus group is causing some controversy.

Liu et al. found that determining the sequence of the 16S rRNA gene did not provide sufficient information to be able to differentiate the bacteria belonging to the B. cereus group, because these bacteria are highly conservative. The authors also noted that the presence or absence of cry genes in B.thuringiensis bacteria cannot be a phenotypic characteristic that differences them from other bacteria of the Bacillus group. However, according to the debate about the difference in the species of the bacteria B.anthracis, B.cereus and B.thuringiensis, can be achieved using the analysis of the whole genome sequence. They have shown that the housekeeping genes pycA and ccpA are suggested to identify rapidly of this group isolates [26].

Indeed, the data we obtained showed that the 16S rDNA gene sequences were not genetically distinct from bacteria of the B. cereus group in the phylogenetic tree. The 16S rRNA gene nucleotide sequences of native Bt strains were found to be 99% similar to other Bt strains that were investigated. However, these local strains are very similar to other strains of the Bacillus group, and it is difficult to separate them into different species from a molecular-genetic point of view.

We identified these strains as being of the B.thuringiensis species only by having of bipyramidal and also the presence of cry-genes. In future, we plan to conduct a study to compare the number of 16S rDNA gene repeats, using pycA and ccpA genes by whole-genome sequencing.

Modern studies show that the population variability of bacteria is related to the processes of internal genome recombination in the genetic material of the cell [27]. It is known that the population of microorganisms is heterogeneous and is characterized by the presence within the population (of one strain) of cells that differ with regard to a number of colonial-morphological and physiological-biochemical properties and are capable of dissociative transitions with high frequencies (10-2-10-4 per cell division) [28].

We can observe in our study that such variability has changed the genetic characteristics of the bacterium B.thuringiensis in one population environment. In the studies conducted by G. Vilas-Boas et al., five strains of B.thuringiensis that were isolated from different population environments (F and S sites) were distinguished by their electrophoretic types (ETs) and numbers of alleles at the gene locus [29]. The authors identified isolates from different ecological zones of Brazil and found that these were genetically differentiated according to random amplified polymorphic DNA (RAPD) PCR results. They also found that the plasmid DNA profile of the two isolates from the granary was different [30].

CONCLUSION

The results of our research show that although strains Btl, Btl8fo, Bt26 and Bt3l were isolated from one population zone around Kibrai, in the Tashkent region, they showed genetic differences. This may be because of the rapid genetic changes that occur in Bt strains within a population zone that is under the influence of external factors or as a result of changes in the host organisms. Some of these eight Bt strains were found to contain specific cry genes. The strains were differentiated because Bt84 strain had cry9Ba-I and crylAc genes, while the Bt9l strain had crylAb and cry9Ba-I genes. Both Bt93 and Bt94 strains were found to have the same crylAb and cry9Ba-I. Therefore, the wide distribution of B.thuringiensis bacteria throughout the world and the fact that these bacteria live in different ecological environments and are influenced by biotic and abiotic factors can lead to a high level of genetic variability.

ACKNOWLEDGMENTS

This research was financially supported by Ministry of Innovative Development of the Republic of Uzbekistan, Tashkent. The researchers are appreciative for the financial support.

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