CLONING AND CHARACTERISATION OF cDNA ENCODING LEGUME SEED DEFENSIN AND ITS ACTIVITY AGAINST BRUCHIDS
Loading...
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Defensins 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.