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

Received: 8 May 2023/Accepted: 18 October 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 2^nd 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

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*Corresponding author; email: fakhar.yunus@lcwu.edu.pk