BACILLUS THURINGIENSIS 1FO SHTAMMINING KO'SAK QURTIGA FAOLLIGI VA MOLEKULAYAR XARAKTERISTIKASI Текст научной статьи по специальности «Биологические науки»

BACILLUS THURINGIENSIS 1FO SHTAMMINING KO'SAK QURTIGA FAOLLIGI VA MOLEKULAYAR XARAKTERISTIKASI Qobilov Fazliddin

O'zRFA Mikrobiologiya instituti, Toshkent, O'zbekiston.

Email: fazliddinbiology@gmail.com https://doi.org/10.5281/zenodo. 8372635

Annotatsiya. Mazkur tadqiqot ishida, B.thuringiensisning yangi tabiiy shtammining molekulyar genetik identifikatsiyasi v a Lepidoptera oilasiga mansub zararkunanda hasharotlarga qarshi faolbo'lgan cry (kristal) va vip (vigetativ insektitsid oqsil) oqsillarini kodlovchigenlarni aniqlash haqida ma'lumotlar keltirilgan. Bunda, 16S rDNK identifikatsiyasi bo'yicha mazkur shtamm B.thuringiensis turiga yaqin ekanligi aniqlandi va B.thuringiensis 1fo shtammi deb nomlandi. Bundan tashqari, Bt1fo shtammida cry1 oilasining cry1Aa, cry1Ac va vip3 genlari, hamda cry2 oilasi ham uchrashligi tajribalar davomida aniqlandi va bu genlar Lepidopterian hasharotlarga qarshi faollikka ega bo'lgan crylAa, crylAc va vip3A oqsillarini kodlashi adabiyotlardan ma'lum bo'ldi. Shuningdek, Btlfo shtammiHelicoverpa armigera hasharotining 2-3 yoshli lichinkalariga qarchi faolligi o'rganilganda 7 kun ichida 100% lichinkalar nobud bo'lganligi laboratoriya tajribalarida qaydetildi.

Kalit so'zlar: Bacillus thuringiensis, identifikatsiya, cry, vip genlari, Lepidoptera, S-endotoksinlar.

Abstract. In this study, molecular genetic identification of a new native strain of B.thuringiensis and identification of genes encoding cry (crystal) and vip (vegetative insecticidal protein) proteins active against harmful insects belonging to the Lepidoptera family are presented. In this case, according to 16S rDNA identification, this strain was found to be close to B.thuringiensis species and was named B.thuringiensis lfo strain. In addition, in the Btlfo strain, cry1Aa, cry1Ac, and vip3 genes of the cry1 family, as well as the cry2 family, were found during the researches, and it was knownfrom the literature that these genes encode CrylAa, CrylAc, and Vip3A proteins, which have activity against lepidopteran insects. Also, when the activity of the Btlfo strain on 2-3-year-old larvae of Helicoverpa armigera insect was studied, it was noted in laboratory experiments that l00% of the larvae died within 7 days.

Keywords: Bacillus thuringiensis, identification, Cry, Vip genes, Lepidoptera, S-endotoxins.

Аннотация. В работе представлена молекулярно-генетическая идентификация нового природного штамма B. thuringiensis и идентификация генов, кодирующих белки cry (кристаллический) и vip (вегетативный инсектицидный белок), активные в отношении вредных насекомых семейства Lepidoptera. При этом по данным идентификации 16SрДНК этот штамм оказался близким к виду B. thuringiensis и получил название штамма B. thuringiensis lfo. Кроме того, у штамма Btlfo в ходе экспериментов были обнаружены гены crylAa, crylAc и vip3 семейства cryl, а также семейства cry2, причем из литературы известно, что эти гены кодируют crylAa, crylAc и vip3A. белки, обладающие активностью в отношении чешуекрылых насекомых. Также при изучении активности штамма Btlfo на 2-3-летних личинках насекомого Helicoverpa Armigera в лабораторных экспериментах было отмечено, что 100% личинок погибали в течение 7 дней.

Ключевые слова: Bacillus thuringiensis, идентификация, cry, гены vip, чешуекрылые, S -эндотоксины.

Bacillus thuringiensis (Bt) aerob, spora hosil qiluvchi, gram-musbat va entomopatogen bakteriya parasporal kristall oqsillar yoki 5-endotoksinlar (Cry) hosil qiladi. Ushbu Cry oqsillari Lepidoptera, Coleoptera va Diptera kabi hasharotlar zararkunandalari uchun zaharli hisoblanadi [1, 2]. Cry, Cyt va Vip oqsillari tarkibiga asoslanib, har bir shtamm lepidopteran, dipteran, koleopteran yoki hymenopteran zararkunandalariga va hatto kanalar va nematodalar kabi boshqa umurtqasiz hayvonlarga nisbatan faolligi aniqlangan [3, 1, 2]. 1985 yildan 2019 yilgacha 30 yildan ortiq izlanishlardan va skriningdan so'ng, 300 dan ortiq yangi Cry oqsillari aniqlandi va Cry1 dan Cry80 gacha tasniflanadi [4]. Butun dunyoda lepidopteran zararkunandalari har yili qishloq xo'jaligi hosildorligiga katta iqtisodiy zararlarni keltirib chiqaradi [5]. Helicoverpa armígera lichinkalari g'o'zaning novdalari va barglari va kurtaklarini yeb, paxta hosilini kamaytiradi [6]. Yo'qotishlarni kamaytirish uchun asosiy lepidopteran zararkunandalariga qarshi kurashda Bacillus thuringiensisni ifodalovchi g'o'za navlari keng qo'llaniladi [7].

Ko'sak qurti (Helicoverpa armigera) O'zbekistonda asosan Farg'ona, Andijon, Namangan, Toshkent viloyatlari paxta maydonlariga jiddiy zarar yetkazadi. Boshqa viloyatlarda nisbatan zarari kam. Lekin 2020-yila Qashqadaryo viloyatinining Nishon, Mirishkor, tumanlarida ham sezilarli zarar yetkazgan. G'o'zadan tashqari pomidor, makkajo'xori, baqlajon, no'xat va dukkakli ekinlarga juda qattiq zarar yetkazadi Qarshi kurash ishlari o'tkazilmasa 20-30% gacha hosilga zarar yetkazadi. Umuman karshi kurashilmasa 60-80% gacha zararlaydi. O'rtacha qattik zarar keltirgan yillari 30% gacha zarar yetkazadi.

Bt 1fo shtammini Helicoverpa armigera lichinkalariga qarshi sinash uchun avvalo mazkur shtamm Lauria-Bertani (LB) (1L:1% Tryptone, 0.5% Yeast Extract, 1 % NaCl)) suyuq ozuqa muhitiga ekildi. Bt 1fo shtammi spora va kristal oqsillari hosil bo'lgunga qadar tebratkichda 150 ay/daq, 30oC haroratda inkubatsiya qilindi. Spora va kristal hosil qilinishi mikroskopda tekshirib turildi. Yettinchi kunga kelib Bt fo shtammi to'liq spora va kristallar hosil qilgani kuzatildi (1 rasm).

Tayyor Bt 1fo tarkibli preparatni selder o'simligiga sepib, namlantirib Helicoverpa armigera ning 2-3 yoshli lichinkalari oziqlantirildi va yeti kun davomida kuzatildi (6-Rasm).

1-Rasm. B.thuringiensis 1fo shtammining spora va kristal oqsillarining mikroskopik ko'rinishi. A-spora; B-kristal oqsil.

B.thuringiensis 1fo shtammidan umumiy DNK ajratish: Laboratoriya kolleksiyasida mavjud 100 ga yaqin B.thuringiensis shtammlari orasidan Bt1fo shtammi mikroskpik tahlil orqali ya'ni kristal hosil qilishiga qarab tanlab olindi. Mazkur Btlfo shtammining 16S rRNK geni bo'yicha molekulyar genetik identifikatsiyasi va cry, vip genlarini aniqlash uchun genom DNK si ajratish amalga oshirildi. Bunda modifikatsiyalan Marmur [8] usulidan foydalanildi. Buning uchun

Lauria-Bertani ozuqasida 16 soat davomida 37oC xaroratda o'sgan bakteriya kUturalari o'stirildi va DNK ajratildi (1-rasm).

Praymerlar tanlash: Bt1fo shtammining 16S rDNK identifikatsiyasi va cry, vip genlarini aniqlash uchun praymerlar dizayn qilindi. Dizayn qilingan praymerlar SnapGene dasturi yordamida tekshirib ko'rildi va IDT (Integrated DNA Technologies) frmasi tomonidan sintez qilindi (1-Jadval).

1-jadval

B. thuringiensis 1fo shtammidagi cry va vip genlarini aniqlash uchun praymerlar

ilish

Prayme Gen nomi Havolala

rlar Praymerlar ketma-ketligi J/A r

nomi

cry1F CATGATTCATGC GGCAGATAA AC cry1 274277

cry1R TTGTGACACTTC TGCTTCCCA TT

cry2F GTTATTCTTAATGCAGATGAA TGGG cry2 689701

cry2R CGGATAAAATAATC TGGGAA ATAGT [9]

cry3F CGTTATCGCAGAGAGATGAC Cry3Aa 589-

ATTAAC , -Ba,-Bb, -Ca 604

cry3R CATCTGTTGTTTC TGGAGGCA AT

cry4F GCATATGATGTAGC GAAACA AGCC cry4 439

cry4R GCGTGACATACCCATTTCCAG GTCC

cry7-8F AAGCAGTGAATGCCTTGTTTA C cry7Aa, -Ab; 420423

cry7-8R C TTC TAAAC C TTGAC TAC TT cry8Aa, -Ba, -

Ca

Lep1A CCGGTGCTGGATTTGTGTTA cry1Aa, cry1Ab 490

Lep1B AATCCCGTATTGTACCAGCG cry1Ac

Dip1A CAAGCCGCAAATCTTGTGGA cry4A, cry4B 797

Dip1B ATGGCTTGTTTCGCTACATC [10]

Col1A GTC C GC TGTATATTC AGGTG cry3A 649

Col1B C AC TTAATC C TGTGAC GC C T

5- GGATC C GATGAAAAATAT GA vip1A 2300

vip1A 3- vip1A AGAA GTC GAC TTATC TAGATTTGTT AGGT [ 1 1 ]

5- GGATCCGATGAAAAGAATGG vip2A 1300

vip2A AGGG

3- GTCGACTTAATTTGTTAATAA

vip2A TGTTG

vip3AF vip3AR ATGAACAAGAATAATACTAA A GC GGC C GC TTAC TTAATAGAG AC vip3A 2300 [ 1 2]

B. thuringiensis 1fo shtammining 16S rRNK, Cry va Vip genlarining PZR amplifikatsiyasi: PZR amplifikatsiyasini amalga oshirishda universal oligonukleotid praymerlardan foydalanild i: 27F (AGAGTTTGATCMTGGCTCAG) va 1492R (GGTTACCTTGTTACGACTT) [13]. Bakteriya shtammidan ajratilgan DNK na'munalari PZR-amplifikatsiyasi GenPak® PCR MasterMix kitida amalga oshirildi. Bunda, reaksiya umumiy 20 mkl miqdorida tayyorlanib, 10 mkl Dilution, 8,2 mkl ikki marta distillangan suv, 0,4 mkldan (27F va 1492R) praymer va 1 mkl DNK namunalaridan tarkib topdi. PZR amplifikatsiya optimizatsiyasi boshlang'ich denaturatsiya 94°C 3 daqiqa, denaturatsiya 94°C 40 soniya, odjig (primer annealing) 55°C 40 sekund, elongatsiya 70°C 90 soniya, yakuniy elongatsiya 70°C 10 daqiqa, davomida takroriy 35 siklda olib borildi. Amplikonlar etidium-bromid bilan bo'yalgan 2 % li agarozali gelda elektroforez yordamida deteksiya qilindi (2A-rasm).

B. thuringiensis 1fo shtammidagi cry va vip genlarini birlamchi PZR skriningi uchun 2x Green PCR Master Mix majmuasidan foydalanildi. Bu an'anaviy Taq DNK polimeraza bilan solishtirganda yuqori sezuvchanlik, uzunroq PCR mahsulotlari va yuqori rentabellikni ta'minlaydi (Thermo Fisher). Hamda, tadqiqotning keying bosqichlari ya'ni sekvins va klonlash usullarini amalga oshirish uchun genlarni to'liq sintez bo'lishini ham ta'minlaydi. Bunda PZR reaksiya umumiy 10 mkl ga tayyorlanib: 2x Green PCR MM-5mkl, ikki marta distillangan suv-3.4 mkl, forward-rewerse praymerlar (1-Jadval) 0.3 mkl dan va shtamm genom DNK-1 mkl ni tashkil qildi. PZR amplifikatsiyadan chiqgan na'munalari 0.7% li agaroza gelida ko'rildi (2B-rasm).

Sekvenslash uchun PZR mahsulotlari 2% agaroza gelidan steril skalpel yordamida kesib olindi va QIAquick® Gel tozalash to'plami yordamida tozalandi [14]. Tozalangan PZR mahsulotlarining miqdori NanoDrop apparatida o'lchandi (NanoPhotometer® N60; IMPLEN). PZR mahsulotlari Rossiyaning СИНТОЛ frmasi tomonidan sekvens qilindi.

Bt1fo shtammining 16S rRNK, cry1Aa va cry1Ac genlarining ketma-ketlik natijalari NCBI BLAST ma'lumotlar bazasidagi turlar bilan o'zaro solishtirildi. (http://www. ncbi.nlm. nih. gov/BLAST). Bt1fo shtammining 16S rRNK, cry1Aa va cry1Ac genlarining ketma-ketlik natijalari NCBI BLAST ma'lumotlar bazasidagi turlar bilan o'zaro solishtirildi. B.thuringiensis 1fo shtammining 16S rRNK, cry1Aa va cry1Ac genlariga asoslangan filogenetik shajaralari MEGA-X dasturi orqali tuzildi. (version 10.1.8) [15].

Laboratoriya kolleksiyasidan tanlab olingan B.thuringiensis 1fo shtammi ustida tadqiqotlar olib borildi. Birinchi navbatda, mazkur shtammning o'ziga xos bo'lgan kristall oqsil siztez qilishi mikroskopik tahlilda namoyon bo'ldi. O'z navbatida bu kristall oqsillar turli xil xasharotlarga toksiklik xususiyatlarini namoyon qiladi. Shundan so'ng, Btlfo stammining 16S rDNK geni bo'yicha identifikatsiya qilish, cry va vip genlarini aniqlash uchun birlamchi PZR tahlilini amalga

oshirish uchun jami 24 ta praymerlar dizayn qilindi va siztez qilinib PZR amplifikatsiyasi amalga oshirildi. Bunda dastlab modifikatsiyalangan Marmur usuli yordamida Bt1fo shtammidan genom DNK si ajratildi (lA-rasm).

2A-pacM. B.thuringiensis shtammining genom DNKsi.

1fo

2B-pacM.

B.thuringiensis 1fo

shtammining 16S rDNK geni PZR maxsuloti.

2A-rasmda ko'ringanidek, B.thuringiensis 1fo shtammidan yuqori sifatli va toza genom DNK modifikatsiyalangan Marmur usuli yordamida ajratildi. 2B-rasmda ushbu bakteriyaning 16S rDNK genining taxminan 1500 bp hosil bo'ldi. Ushbu Bt 1fo ning 16S rRNK genining sekvenslangan mahsuloti 1443 bp, cry1Aa geni 1842 bp va cry1Ac geni esa 1465 bp bo'lib, ushbu genlar NCBI BLAST ma'lumotlar bazasidagi bakteriya turlarining 16S rRNK va cry genlari bilan o'zaro solishtirildi. Natijada, shtammning 16S rRNK geni tahliliga ko'ra mazkur shtamm B.bombysepticus, B.cereus, B.subtilis, B.thuringiensis kabi turlar bilan 99,51% o'xshash ekanligi aniqlandi.

Shundan so'ng, Mega4 bioinformatik dasturining Maximum-likelihood statistik usuli yordamida Bt1fo shtammining 16S rRNK, cry1Aa va cry1Ac genlariga asoslanib filogenetik shajaralari tuzib chiqildi. Bt1fo shtammi 16S rDNK geni bo'yicha B.toyonensis, B.bombysepticus, B.cereus, B.subtilis, B.thuringiensis turlari bilan o'zaro solishtirildi (3-rasm).

0.0000 0.0000

0.0003 0.0015

~|o.OOL

0.0000 0.0022

■s

LC60295 5.1 Bacillus toy im eu s is \Iaii8.04-2316S iR.NA geuepailialsequeuce A B677944.1 Bacillus thuringiensis strain RG17-1116S iKNA gene partial sequence OP520898.1 Bacillus hombysepticus strain FPRC23 16S rKNA geue partial sequence OP520899.1 Bacillus Ь oruby septicus straiu FPRC30 16S rKNA geue partial sequence A B617475.1 В а с illu s t li u rin gien sis serovar kurstaki 16 S rKNA gene partial sequence GQ202000.1 B.thuiingimsisserovar kurstakistrain AR-14ItiS rKNA geuepailialsequeuce OU707432.1 Bacillus toyonensis isolate Cll paitial 16S rKNA geue B.thuringiensis strain lfo 16S rKNA gene partial sequence LC178545.1 Bacillus tliuringieusis isolate: X-l 16S rKNA gene paitial sequence LT603028.1 Bacillus cereusstrain ISUWYZÖ4 16S rKNA geue partial sequeuce OM108144.1 Bacillus tropicus strain CU96 16S rKNA gene paitial sequence OR195851.1 Bacillus tropicus strain AG13116S rRXA geue paitial sequence 3-\Т745970.1 Bacillus cereus straiu SGC isolate 2 16S rKNA geue paitial sequeuce

0.0046

I-AB192294.2 Bacillus subtilis 16S rKNA geuepailialsequeuce

0.0296 С17854 6.1 Bacillus sutitilis isolate X-2 16S rKNA geue paitial sequeuce

3-Rasm. B.thuringiensis 1foshtammining 16S rDNK geni bo'yicha tuzilgan filogenetik shajarasi.

Mega4 bioinformatik programmasining Maximum-likelihood statistik usulidan foydalanildi. B.thuringiensis 1fo shtammining 16S rDNK geni filogenetik shajarasi ko'ra, mazkur shtamm B.cereus va boshqa turlardan farq qilishi, hamda B.thuringiensis turi bilan bir xil ekanligi aniqlandi. Adabiyotlardan ma'lumki, universal primerlarga asoslangan 16S rDNK gen ketma-ketligi B.cereus va B.thuringiensis o'rtasida yuqori o'xshashlikni ya'ni 99% ni ko'rsatadi [16]. Bu o'xshashlikka qaramay, B.thuringiensis turlari o'ziga xos bo'lgan kristall va Vip oqsillarni kodlovchi genlari bilan B.cereus va boshqa turlar bilan ajralib turadi [17].

So'ngra, Btlfo shtammida cry va vip oilasiga tegishli genlarni aniqlash maqsadida PZR amplifikatsiyasi amalga oshirildi. Bunda, cry1-8 va vip1, 2, 3 oilasi genlarini aniqlovchi praymerlardan foydalandi.

4-rasm. Bt 1fo shtammining cry va vip genlarining birlamchi PZR tahlili: M-1kb DNK marker. 1- cry1, 2- cry2, 3- cry3, 4- cry4, 5- cry7-8, 6- Lep1, 7- Dip1, 8-cry3A, 9- vip1A, 10- vip2A, 11- vip3A, ММ-Manfiy nazorat.

4-rasmdan ko'rinib turibdiki, Bt 1fo shtammida cry1, cry2 va Vip3 oilalariga tegishli bo'lgan Cry va Vip genlari mavjudligi aniqlandi. 6-qatordagi Lep1 praymerlari hosil qilgan PZR maxsuloti esa mazkur shtammda cry1 oilasining cry1Aa, cry1Ab va cry1Ac genlari borligini yana bir bor tasdiqladi. Shuningdek, Bt1fo shtammida cry3, cry4, cry7-8 va vip1A, vip2A genlari uchramasligi birlamchi PZR tahliliga ko'ra tasdiqlandi.

cry1Aa va cry1Ac genlari esa NCBI BLAST ma'lumotlar bazasidagi cry1Aa va cry1Ac genlar bilan 100% o'xshash ekanligi namoyon bo'ldi. Mazkur Bt 1fo shtammini kristal oqsillarini hosil qilishi va ushbu oqsillarni kodlovchi bir nechta genlari uchrashligiga qarab, bu shtamm Bacillus thuringiensis ekanligi tasdiqlandi va biz B.thuringiensis 1fo deb nomladik.

So'ngra, Mega4 bioinformatik dasturining Maximum-likelihood statistik usuli yordamida Bt1fo shtammining cry1Aa va cry1Ac genlariga asoslanib filogenetik shajarasi tuzib chiqildi. Bt1fo shtammining cry1Aa va cry1Ac genlari bo'yicha NCBI bazasidan cry1A oilasining cry1Aa, cry1Ab va cry1Ac genlari bilan taqqoslandi va filogenetik shajaralari tuzildi. cry2A geni esa outgroup sifatida tanlandi (5-rasm).

Filogenetik shajarasidan ko'rinib turibdiki, B.thuringiensis lfo shtammining cry1Aa va cry1Ac genlari NCBI BLAST natijasi bilan bir xil ko'rsatkichni ya'ni bu ikkla gen ham cry1Aa va cry1Ac genlariga ta'luqli ekanligi tasdiqlandi. Bunda, Bt1fo stammining cry1Aa geni 100% o'xshashlik bilan filogenetik daraxtning cry1Aa guruhi ichida joylashgani va xuddi shu ko'rsatkich bilan Btlfo stammining cry1Ac geni 100% o'xshashlik bilan filogenetik daraxtning cry1Aa guruhi ichida joylashgani namoyon bo'ldi. Adabiyotlardan ma'lumki, B.thuringiensis serovar kurstaki HD1 shtammida beshtagacha cry genlari (cry1Aa, cry1Ab, cry1Ac, cry2Aa ва cry2Ab) uchrashligi aniqlangan [18].

100% i— KT900180.1 Bacillus thuringiensis strain 1905 Cry1Ac39 gene partial sequence cds

100%

100% 100% 100%

100% 100% 100%

100% 100% 100%

Bacillus thuringiensis 1fo strain CrylAc gene partial sequence

JQ819250.1 Bacillus thuringiensis strain 2B crylAc protein gene partial sequence cds AY730621.1 Bacillus thuringiensis plasmid CrylAc gene partial sequence cds EU282379.1 B.thuringiensis serovar kurstaki strain W015 Cry1Ac22 gene partial sequence cds MK184463.1 Bacillus thuringiensis strain ABTS-351 Cry1Ac gene partial sequence cds KT900185.1 Bacillus thuringiensis strain 2122 Cry1Ab36 gene partial sequence cds KT900179.1 Bacillus thuringiensis strain 1905 Cry1Ab35 gene partial sequence cds AF358861.1 Bacillus thuringiensis crystal endotoxin Cry1Ab (cry1Ab) gene partial sequence I— AF254640.1 Bacillus thuringiensis insecticidal protein P (cry1Ab) gene partial sequence cds '%'— X54939.1 B. thuringiensis cryIA(b) gene partial sequence -Bacillus thuringiensis 1fo strain Cry1Aa gene partial sequence

-MK391629.1 Bacillus thuringiensis strain MPU B5 Cry1Aa gene partial sequence cds

-KF938682.1 Bacillus thuringiensis strain HTS-S-38 Cry1Aa gene partial sequence cds

-KT900178.1 Bacillus thuringiensis strain 1905 Cry1Aa25 gene partial sequence cds

ioo% |— MK184475.1 Bacillus thuringiensis strain ABTS-1857 Cry1Aa gene partial sequence cds

ioo^L HQ685121.1 Bacillus thuringiensis strain LS-R-21 Cry1Aa gene partial cds -DQ020578.1 Bacillus thuringiensis Cry2Ac gene partial sequence cds

5-rasm. B.thuringiensis lfo shtammining cry1Aa va cry1Ac genlarining filogenetik shajarasi

Bizning Bt1fo shtammimizda esa cry1 oilasiga tegishli cry1Aa va cry1Ac genlari va vip3A geni mavjud ekanligi tadqiqotlarimiz natijasi namoyon bo'ldi. Bundan tashqari, mazkur shtammda cry2 oilasi genlari ham borligi aniqlangan bo'lib, keying tadqiqotlarimizda bu oila genlarini va boshqa genlarni aniqlash ustida tadqiqotlarimizni davom ettiramiz.

B.thuringiensis 1fo shtammining ko'sak qurti lichinkalariga qarshi faolligini baholash: Bt1fo asosli biopreparati (Spora va kristalli) ko'sak qurti lichinkalariga selder o'simligiga sepib oziqlantirildi. Tajribada 50 dona 2-3 yoshli lichinkalardan foydalanildi va bir hafta davomida kuzatildi. Nazoratda esa 10 ta shu yoshdagi lichinkalar kuzatildi (6-rasm).

100%

100%

ISSN: 2181-3337 | SCIENTISTS.UZ

INTERNATIONAL SCIENTIFIC JOURNAL SCIENCE AND INNOVATION

ISSUE DEDICATED TO THE 80TH ANNIVERSARY OF THE ACADEMY OF SCIENCES OF THE REPUBLIC OF UZBEKISTAN

6-Rasm. A- Helicoverpa armigera hasharotining sog'lom lichinkasi, B-B.thuringiensis 1fo asosli praparatdan nobud bo'lgan Helicoverpa armigera lichinkasi.

Tajribaning 7 kuniga kelib Btlfo asosli biopreparat bilan oziqlantirilgan 50 dona ko'sak qurti lichinkalarining barchasi (100%) nobud bo'lganligi kuzatildi. Nazoratdagi lichinkalar esa 10 kunga kelib barchasi (100%) g'umkaka ketdi va 23-26 kunda urug'landi.

Hozirgi kunda o'simliklarni zararkunanda hasharotlardan himoya qilishning eng samarali yo'llardan bin biologik agentlardan foydalasnishdir. Biz tadqiqot olib borgan B.thuringiensis 1fo bakteriyasi ham xuddi shunday samarali shtammlardandir. Tadqiqotlar davomida mazkur shtamm 16S rDNK geni bo'yicha molekulyar genetik identifikatsiya qilindi. Uning birlamchi PZR tahlili va sekvens natijalariga ko'ra, Bt 1fo shtammida cryl oilasining crylAa, crylAc va vip3A genlari mavjudligi, hamda cry2 oilasi genlari ham uchrashligi aniqlandi va ushbu genlar NCBI ma'lumotlar bazasidagi cry va vip genlari bilan o'zaro taqqoslanib, filogenetik daraxtlari tuzib chiqildi. Shundan so'ng, Btlfo shtammini Helicoverpa armigera lichinkalariga nisbatan faolligi o'rganilganda. Btlfo asosli preparat bilan oziqlantirilganda Helicoverpa armigera ning 2-3 yoshli lichinkalari 7 kunga kelib 100% nobud bo'lganligi kuzatildi. Btlfo shtammini qishloq xo'jaligi ekinlarini Helicoverpa armigera kabi hasharotlardan himoya qilish uchun samarali bioagent sifatida foydalanish mumkin. Bt1fo shtammida mavjud cry1Aa, cry1Ac va vip3A genlarini mavjudligi, yaqin kelajakda transgen o'simliklarini yaratish imkoniyatlarini beradi.

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