Journal of Environmental Biology

Pheno-molecular confirmation and protein structure analysis of Wild Vs CRISPR white mutant Bactrocera dorsalis

S. Dash^1,2, R. Asokan³ and J.D. Naik¹*     

¹Department of Agricultural Entomology, College of Agriculture, UAS, GKVK, Bengaluru-560 065, India

²Division of Entomology, ICAR-Indian Agricultural Research Institute, Pusa campus, New Delhi-110 012, India

³Division of Basic Sciences, ICAR-Indian Institute of Horticultural Research, Bengaluru-560 089, India

Received: 29 August 2024                   Revised: 02 January 2025                   Accepted: 14 February 2025

*Corresponding Author Email : djnent98@gmail.com                     *ORCiD: https://orcid.org/0000000336062580                     

Abstract

Aim: This investigation aimed for embryonic G0 microinjection of ribonucleoprotein (RNP) complex (sg360+Cas9) for restriction of white melanin synthesising gene in Bactrocera dorsalis followed byphenotypic and molecular confirmation and 2D and 3D protein structure comparison of wild Vs CRISPR white mutant.

Methodology: White gene responsible for melanin pigmentation was cloned followed by synthesis and in vitro restriction analysis of designed sgRNA (sg360). Microinjection was targeted in G0 embryosfollowed by phenotypic characterisation and molecular validation. Comparative analysis of amino acid sequences, 2D and 3D structure of wild Vs CRISPR white mutant was done using ClustalW and Phyre2 respectively.

Results: Microinjected RNP complex restricted white gene leading to generation of white eyed mutants. Amino acid alignments showed significant frame shift (valine substituted for tryptophan) and introduction of stop codons in the mutant hindering white protein production. Predicted 2D protein structure showed more α-helix in the mutant (72%) compared to the wild (22%). Similarly, β-sheets were nullified and major deletions in the3D protein structure were observedin mutant.

Interpretation: The study validated variation in amino acids alignments and 2D and 3D protein structure and conformation, leading to production of non-functional white protein in B. dorsalis.

Key words: Bactrocera dorsalis, CRISPR/Cas9, Microinjection, White protein

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