Sains Malaysiana 52(11)(2023): 3061-3073
http://doi.org/10.17576/jsm-2023-5211-04
Random Mutagenesis to Enhance the Toxicity
of Bacillus thuringiensis Cry Proteins against Earias vittella (F.)
(Mutagenesis Rawak untuk Meningkatkan Ketoksikan Protein Cry Bacillus thuringiensis terhadap Earias vittella (F.))
HAFSA ZAHEER, HUMA KHURSHID,
FAKHAR-UN-NISA YUNUS*, FARKHANDA MANZOOR, GHAZALA JABEEN & ZAKIA KANWAL
Lahore College for Women University, Lahore,
Pakistan
Diserahkan: 8 Mei 2023/Diterima: 18 Oktober 2023
Abstract
Insecticidal proteins derived from Bacillus thuringiensis (Bt) are widely utilized in a variety of insect control
applications,including sprays and transgenic crops.The development of
resistance in pests, on the other hand, can lessen the effectiveness of Bttoxins. In this study, we made efforts to enhance the toxicity of two cry proteins Cry1Ac and
Cry2Aa through random mutagenesis against cotton bollworm (Earias vitella), one of the most
destructive cotton pests in Pakistan. Random mutagenesis is an important tool for
elucidating protein structure-function relationships and for modifying proteins
to enhance or change their characteristics. We focused on whole cry proteins
for random mutagenesis througherror-prone
PCR and constructed a recombinant library of cry proteins. Sequence analysis of eight mutants showed the
mutations of 34 different nucleotides in Cry1Ac and Cry2Aa genes. All mutants
were spared for toxicity bioassays against 2nd instar larvae of
spotted bollworm. Cry1Ac mutant RM1AcM4 (D242E) and Cry2Aa mutants RM2AaM2
(T354A, T492R, F511L, G585E, D606Y) showed enhanced toxicity as compared to
proteins without mutation. These two mutants comprise the mutations in
domain-II of cry proteins important
in specificity determining regions on midgut receptors in insect pests.
Keywords: Bacillus thuringiensis; error-prone PCR; random mutagenesis; Earias vitella
Abstrak
Protein racun serangga yang diperoleh
daripada Bacillus thuringiensis (Bt) digunakan secara meluas dalam
pelbagai aplikasi kawalan serangga, termasuk semburan dan tanaman transgenik.
Perkembangan rintangan pada perosak sebaliknya boleh mengurangkan keberkesanan
toksin Bt. Dalam kajian ini, kami
berusaha untuk meningkatkan ketoksikan dua protein cry Cry1Ac dan Cry2Aa melalui mutagenesis rawak terhadap ulat bulu
kapas (Earias vitella), salah satu
daripada perosak kapas yang paling teruk di Pakistan. Mutagenesis rawak ialah
alat penting untuk menjelaskan hubungan struktur-fungsi protein dan untuk
mengubah suai protein untuk meningkatkan atau mengubah cirinya. Kami memberi
tumpuan kepada keseluruhan protein cry untuk mutagenesis rawak melalui PCR yang terdedah kepada ralat dan membina
perpustakaan rekombinan protein cry.
Analisis jujukan lapan mutan menunjukkan mutasi 34 nukleotida berbeza dalam gen
Cry1Ac dan Cry2Aa. Semua mutan telah dikecualikan untuk bioasai ketoksikan
terhadap larva instar kedua ulat bulu. Mutan Cry1Ac RM1AcM4 (D242E) dan mutan
Cry2Aa RM2AaM2 (T354A, T492R, F511L, G585E, D606Y) menunjukkan ketoksikan yang
dipertingkatkan berbanding dengan protein tanpa mutasi. Kedua-dua mutan ini
terdiri daripada mutasi dalam domain-II protein cry yang penting dalam kekhususan menentukan kawasan pada reseptor
usus tengah pada perosak serangga.
Kata kunci: Bacillus thuringiensis; Earias vitella; mutagenesis rawak; PCR terdedah ralat
RUJUKAN
Adams, T.T.,
Eiteman, M.A. & Hanel, B.M. 2002. Solid state fermentation of broiler
litter for production of biocontrol agents. Bioresource Technology 82(1): 33-41.
Bleisch, R.,
Freitag, L., Ihadjadene, Y., Sprenger, U., Steingröwer, J., Walther, T. &
Krujatz, F. 2022. Strain development in microalgal biotechnology - random
mutagenesis techniques. Life 12(7): 961.
de Oliveira, J.A.,
Negri, B.F., Hernández-Martínez, P., Basso, M.F. & Escriche, B. 2023.
Mpp23Aa/Xpp37Aa insecticidal proteins from Bacillus
thuringiensis (Bacillales: Bacillaceae) are highly toxic to Anthonomus grandis (Coleoptera:
Curculionidae) Larvae. Toxins 15(1): 55.
Gould, F.,
Martinez-Ramirez, A., Anderson, A., Ferre, J., Silva, F.J. & Moar, W.J.
1992. Broad-spectrum resistance to Bacillus
thuringiensis toxins in Heliothis
virescens. Proceedings of the National Academy of Sciences 89(17): 7986-7990.
Jan, M.T., Abbas,
N., Shad, S.A. & Saleem, M.A. 2015. Resistance to organophosphate,
pyrethroid and biorational insecticides in populations of spotted bollworm, Earias vittella (Fabricius)(Lepidoptera:
Noctuidae), in Pakistan. Crop Protection 78: 247-252.
Jenkins, J.L., Lee,
M.K., Valaitis, A.P., Curtiss, A. & Dean, D.H. 2000. Bivalent sequential
binding model of a Bacillus thuringiensis toxin to gypsy moth aminopeptidase N receptor. Journal of Biological
Chemistry 275(19):
14423-14431.
Jurat-Fuentes,
J.L., Heckel, D.G. & Ferré, J. 2021. Mechanisms of resistance to
insecticidal proteins from Bacillus
thuringiensis. Annual Review of Entomology 66: 121-140.
Kang, J.N., Roh,
J.Y., Shin, S.C., Koh, S.H., Chung, Y.J., Kim, Y.S., Wang, Y., Choi, H., Li,
M.S., Choi, J.Y. & Je, Y.H. 2007. Dual insecticidal activity of
spodoptera-toxic Bacillus thuringiensis strain transformed with Lepidopteran-specific cry toxin. Journal of
Asia-Pacific Entomology 10(2):
137-143.
Koppenhöfer, A.M.,
Wilson, M., Brown, I., Kaya, H.K. & Gaugler, R. 2000. Biological control
agents for white grubs (Coleoptera: Scarabaeidae) in anticipation of the
establishment of the Japanese beetle in California. Journal of Economic
Entomology 93(1):
71-80.
Lenug, D.W. 1989.
A method for random mutagenesis of a defined DNA segment using a modified
polymerase chain reaction. Technique JMCMB 1: 11-15.
Liao, C., Heckel,
D.G. & Akhurst, R. 2002. Toxicity of Bacillus
thuringiensis insecticidal proteins for Helicoverpa
armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton. Journal of
Invertebrate Pathology 80(1):
55-63.
Lutz, S. &
Patrick, W.M. 2004. Novel methods for directed evolution of enzymes: Quality,
not quantity. Current Opinion in Biotechnology 15(4): 291-297.
Mannion, C.M.,
McLane, W., Klein, M.G., Moyseenko, J., Oliver, J.B. & Cowan, D. 2001.
Management of early-instar Japanese beetle (Coleoptera: Scarabaeidae) in
field-grown nursery crops. Journal of Economic Entomology 94(5): 1151-1161.
Manoj Kumar, A.S.
& Aronson, A.I. 1999. Analysis of mutations in the pore-forming region
essential for insecticidal activity of a Bacillus
thuringiensis δ-endotoxin. Journal of Bacteriology 181(19): 6103-6107.
McNeil, B.C. &
Dean, D.H. 2011. Bacillus thuringiensis Cry2Ab is active on Anopheles mosquitoes: Single D block exchanges reveal critical residues involved in
activity. FEMS Microbiology Letters 325(1): 16-21.
Morse, R.J.,
Yamamoto, T. & Stroud, R.M. 2001. Structure of Cry2Aa suggests an
unexpected receptor binding epitope. Structure 9(5): 409-417.
Naqvi, R.Z., Asif,
M., Saeed, M., Asad, S., Khatoon, A., Amin, I., Mukhtar, Z., Bashir, A. &
Mansoor, S. 2017. Development of a triple gene Cry1Ac-Cry2Ab-EPSPS
construct and its expression in Nicotiana benthamiana for insect
resistance and herbicide tolerance in plants. Frontiers in Plant
Science 8: 55.
Rasila, T.S.,
Pajunen, M.I. & Savilahti, H. 2009. Critical evaluation of random
mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and
hydroxylamine treatment. Analytical Biochemistry 388(1): 71-80.
Reisig, D.D.,
Huseth, A.S., Bacheler, J.S., Aghaee, M.A., Braswell, L., Burrack, H.J.,
Flanders, K., Greene, J.K., Herbert, D.A., Jacobson, A. & Paula-Moraes,
S.V. 2018. Long-term empirical and observational evidence of practical Helicoverpa zea resistance to cotton
with pyramided Bt toxins. Journal of Economic Entomology 111(4): 1824-1833.
Romero, P.A. &
Arnold, F.H. 2009. Exploring protein fitness landscapes by directed evolution. Nature
Reviews Molecular Cell Biology 10(12):
866-876.
Saraswathy, N.
& Kumar, P.A. 2004. Protein engineering of delta-endotoxins of Bacillus thuringiensis. Electronic
Journal of Biotechnology 7(2):
178-188.
Sena da Silva,
I.H., Gómez, I., Pacheco, S., Sánchez, J., Zhang, J., Luque Castellane, T.C.,
Aparecida Desiderio, J., Soberón, M., Bravo, A. & Polanczyk, R.A. 2021. Bacillus thuringiensis Cry1Ab domain III
β-16 is involved in binding to prohibitin, which correlates with toxicity
against Helicoverpa armigera (Lepidoptera: Noctuidae). Applied and Environmental Microbiology 87(2): e01930-20.
Shan, S., Zhang,
Y., Ding, X., Hu, S., Sun, Y., Yu, Z., Liu, S., Zhu, Z. & Xia, L.
2011. A Cry1Ac toxin variant generated by directed evolution has enhanced
toxicity against Lepidopteran insects. Current Microbiology 62: 358-365.
Shikano, I. &
Cory, J.S. 2014. Genetic resistance to Bacillus
thuringiensis alters feeding behaviour in the cabbage looper, Trichoplusia ni. PLoS ONE 9(1): e85709.
Talaei-Hassanloui,
R., Bakhshaei, R., Hosseininaveh, V. & Khorramnezhad, A. 2014. Effect of
midgut proteolytic activity on susceptibility of lepidopteran larvae to Bacillus thuringiensis subsp. kurstaki. Frontiers in
Physiology 4: 406.
Torres-Quintero,
M.C., Gómez, I., Pacheco, S., Sánchez, J., Flores, H., Osuna, J., Mendoza, G.,
Soberón, M. & Bravo, A. 2018. Engineering Bacillus thuringiensis Cyt1Aa toxin specificity from dipteran to
lepidopteran toxicity. Scientific Reports 8(1): 4989.
Van Dillewijn, P.,
Vílchez, S., Paz, J.A. & Ramos, J.L. 2004. Plant‐dependent active
biological containment system for recombinant rhizobacteria. Environmental
Microbiology 6(1):
88-92.
Van Frankenhuyzen, K. 2009.
Insecticidal activity of Bacillus thuringiensis crystal proteins. Journal of Invertebrate
Pathology 101(1): 1-16.
Vanhercke, T.,
Ampe, C., Tirry, L. & Denolf, P. 2005. Reducing mutational bias in random
protein libraries. Analytical Biochemistry 339(1): 9-14.
Vílchez, S.,
Jacoby, J. & Ellar, D.J. 2004. Display of biologically functional
insecticidal toxin on the surface of λ phage. Applied and
Environmental Microbiology 70(11):
6587-6594.
Wang, F., Liu, Y.,
Zhang, F., Chai, L., Ruan, L., Peng, D. & Sun, M. 2012. Improvement of
crystal solubility and increasing toxicity against Caenorhabditis elegans by asparagine substitution in block 3 of Bacillus thuringiensis crystal protein
Cry5Ba. Applied and Environmental Microbiology 78(20): 7197-7204.
Wei, J., Zhang, Y.
& An, S. 2019. The progress in insect cross‐resistance among Bacillus thuringiensis toxins. Archives
of Insect Biochemistry and Physiology 102(3): e21547.
Yunus, F.N.,
Makhdoom, R. & Raza, G. 2011. Synergism between Bacillus thuringiensis toxins Cry1Ac and Cry2Aa against Earias vitella (Lepidoptera). Pakistan
Journal of Zoology 43: 575-580.
Zhang, N., Liu,
R., Shu, C., Zhang, J., Li, H. & Gao, J. 2013. Construction and analysis of
Vip3A insecticidal protein random recombination library. Biotechnology
Bulletin 3: 160.
Zheng, A., Zhu, J.,
Tan, F., Guan, P., Yu, X., Wang, S., Deng, Q., Li, S., Liu, H. & Li, P.
2010. Characterisation and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain
JF19-2. Annals of Microbiology 60: 129-134.
*Pengarang untuk surat-menyurat; email: fakhar.yunus@lcwu.edu.pk
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