Cloning, expression and characterization of arcelin and its impact on digestive enzymes of the stored product insect pest, Callosobruchus maculatus (F.)

Karuppiah Hilda,Sreeramulu Bhuvaragavan,Sundaram Janarthanan,Mani Meenakumari,Ramakrishnan Kamatchi

doi:10.1016/j.pestbp.2021.104982

Abstract

The pulse beetle Callosobruchus maculatus causes potential damage to legume crops by infesting the seeds, leading to a reduction of total protein content. Arcelin found in the wild accessions of the common bean, is an insecticidal protein that has the potency to hamper the metabolism of the bruchid beetle. The arcelin gene from the wild accession of Phaseolus lunatus was isolated and the ORF encoding 158 amino acids was cloned in pET-45b (+) vector. The recombinant clones were transformed in BL21 STAR (DE3) pLysS cells, and the expressed arcelin was purified using Ni-NTA column. The recombinant protein was used in preparing an artificial diet, and the insecticidal activity was elucidated against the bruchid pest C. maculatus. Adult emergence and seed damage were drastically reduced in the treated groups. The response towards ingested diet by digestive enzymes involved in metabolism was elucidated through quantitative gene expression. The highest expression was observed in the aminopeptidase, followed by upregulation of alpha-amylase, glycoside hydrolase family 31 and cathepsin D-like aspartic protease, and downregulation of cathepsin L-like cysteine protease. The recombinant arcelin demonstrates effective insecticidal activity against the bruchid beetle. The changes in digestive enzymes to counteract the anti-nutritional nature of the protein were the strategies of the insect defense mechanism.

Highlights

Plants have evolved various defense strategies against insect herbivory based on physical barriers, constitutive chemical defenses, and direct and indirect inducible defenses (Berenbaum 1995)
Amplification and sequence analysis of cDNA encoding the arcelin gene Total RNA isolated from seeds of P. lunatus was used as the template for cDNA synthesis
The designed gene-specific primer from the arcelin variants amplified the arcelin gene in the seeds of P. lunatus

Summary

Introduction

Plants have evolved various defense strategies against insect herbivory based on physical barriers, constitutive chemical defenses, and direct and indirect inducible defenses (Berenbaum 1995). The exploitation of the applications of defense proteins has gained importance in the recent past and has substituted the use of synthetic insecticides The utilization of such compounds in crop rotation by conventional plant breeding or genetic engineering has been practiced (Gatehouse et al 1990). Apart from true lectins, lectin-related polypeptides (arcelin/phytohemagglutinin/α-amylase inhibitors) possess insecticidal activity against bruchid pests Among these lectin-related genes, arcelin is an effective insecticidal protein resistant to bruchid beetles and is found in some wild accessions of the common bean, Phaseolus vulgaris. Apart from P. vulgaris, the arcelin-like sequence was reported in seeds of P. acutifolius (Mirkov et al 1994) and P. lunatus (Sparvoli et al 2001) These extensive studies on the arcelin molecule provided insight for designing a gene-specific primer for arcelin, which established the platform for cloning and expression of the arcelin gene from wild seeds of P. lunatus from Indian accession and looking at its impact on digestive enzymes of Callosobruchus maculatus

Methods

Results

Conclusion