Acharjee, SumitaDayma, Jyotsna2024-06-262024-06-262023https://krishikosh.egranth.ac.in/handle/1/5810210908Defensins are small cysteine-rich peptides that play a crucial role in a plant’s innate immune system and are known for their well-established antimicrobial activity. However, their potential insecticidal properties have received limited exploration. Chen and his coworker have identified a defensin protein from mung bean (Vigna radiata) having insecticidal activity against bruchids. Our previous research confirmed the upregulation of a defensin gene in black gram due to the bruchid infestation. In the present study, sequencing of the full-length complementary DNA of the defensin gene from various legumes was performed. We found two amino acid sequence variants, PEP1 and PEP2 with 90 to 97% homology with the previously reported mung bean defensin protein, respectively. The PEP1 variant was found in black gram, pea, cowpea, and common bean, while PEP2 was found in mung bean, chickpea, and pigeon pea. The amino acid sequence alignment showed there are two amino acid substitutions in the signal peptide and three amino acid substitutions in mature protein regions of PEP1 compared to PEP2. Expression analysis of insecticidal defensin in different legumes revealed significant upregulation of the defensin gene in common beans, followed by black gram and cowpea, whereas, downregulation was observed in the case of pea, pigeon pea, mung bean, and chickpea that are susceptible to the bruchid infestation. The signal-truncated defensin protein was expressed in E. coli bacteria with an intein tag and a 32.5kD fusion protein was isolated. The cleavage of the tag through chitin affinity chromatography yielded mature peptides of 5.5 kD. Computational visualization showed that PEP1 displayed stronger interactions with the α-amylase enzyme of bruchids (Callosobruchus sp.), particularly through inter-chain hydrogen bonding when compared with PEP2. Moreover, PEP1 demonstrated higher (>14%) α-amylase inhibition activity compared to PEP2. In the insect bioassays, bruchids fed on PEP1 through an artificial diet at a concentration of 0.2% and 0.3% exhibited no adult emergence compared to PEP2. A plant expression vector was also constructed for expressing the defensin gene in the seeds of legumes. The results suggest the presence of amino acid substitutions in the defensin protein of legumes, which could be associated with the resistance to bruchid infestation. Thus, the expression of the black gram defensin gene regulated by a seed-specific promoter in grain legumes such as chickpea/ pigeon pea could provide durable resistance to bruchids.EnglishThesis