Rekha Kansal

Purification and characterization of trypsin inhibitor from Cicer arietinum L. and its efficacy against Helicoverpa armigera

Protease inhibitors in legumes are one of the most promising weapons that confer resistance against insects by inhibiting proteases present in the gut of insect larvae. In the present study, trypsin inhibitor activity was detected in the seed flour extracts of 10 selected varieties of chickpea. The presence of inhibitor was confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor has been purified to near homogeneity to 60.46 fold and 29.20% recovery from chickpea seeds using heat denaturation, ammonium sulphate fractionation, DEAE-Sephadex A-25 and Sephadex G-75. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 30,000 Da. The purified inhibitor was active over a wide pH range although it retained maximum activity between pH 6 and 10. The inhibitor protein was stable up to 80°C but retained only 40% of activity when heated at 100°C for 20 min. The inhibitor lost its activity completely at 121°C. The chickpea trypsin inhibitor exhibited inhibitory activity against Helicoverpa armigera both in vitro and in vivo. In insect bioassay, a progressive decline in larval weight, growth and survival as well as temporal extension of larval growth was observed after feeding H. armigera larvae on diet supplemented with increasing concentrations of chickpea trypsin inhibitor. The adult emergence was also adversely affected by the inhibitor. It may be concluded that chickpea trypsin inhibitor has insecticidal potential against H. armigera.

Construction of Cowpea (Vigna unguiculata L) cDNA Library and Characterization of an Isolated Lectin Gene

The cDNA library of cowpea was constructed in λ ZAP II vector using poly (A+) RNA from developing seeds of cowpea. Initially, to demonstrate the presence of lectin genes in the cowpea genome, cowpea total genomic DNA was isolated, Southern blotted and hybridized with the heterologous pea lectin cDNA probe. It is observed that there exists a strong homology between lectin genes of cowpea and pea. Hence, cowpea cDNA library was screened for the presence of lectin genes using pea lectin cDNA probe. After primary, secondary and tertiary screening, eight positive clones were obtained. When subcloned into pBluescript, out of these eight clones, only four showed presence of larger cDNA inserts. When the DNA of these positive clones were Southern blotted and hybridized, they showed strong hybridization with the probe DNA. One of the clones was sequenced completely and the nucleotide sequence also showed strong homology with other legume lectin genes.

Characterization of Vicilin Seed Storage Protein of Chickpea (Cicer arietinum L.)

Vicilin, one of the major storage proteins of chickpea (Cicer arietinum L.) was purified and characterized during seed development. Vicilin was purified by zonal isoelectric precipitation followed by chromatography on DEAE-cellulose column. Vicilin on SDS-PAGE resolved into 5 major bands ranging in mol wt from 14 to 66 kD. More heterogenous pattern emerged on isoelectric focussing. This protein had high amount of amides and low amount of sulphur containing amino acids.

Endophytic Microorganisms: Their Role in Plant Growth and Crop Improvement

Abstract Plant endophytes have been studied since the 1940s and are known to undertake different interactions. The word endophyte means “in the plant” (endon Gr., within; phyton, plant). They are ubiquitous in nature and show association with nearly 300,000 plant species that exist on the earth. Each individual plant is host to one or more endophytes. The use of term was argued as most of the microorganisms do not show any symptoms during initial infection. Petrini (1991) added a new dimension in the characterization of endophytic interactions where the absence of macroscopically visible symptoms was considered as “causing no apparent harm.” The term “endophyte” is mostly used to portray bacteria and fungi “asymptomatic” microbial colonization as a balance of antagonisms between host and endophyte at a particular moment ( Hallmann et al., 1997 ; Schulz and Boyle, 2005 , Schulz and Boyle, 2006 ). The short-lived and delicate balance of antagonism depends on the general status of the host, that is, defense mechanism, nutritional status, different environmental factors, and developmental stages of the endophytic partners.

Isolation and in silico characterization of cDNA encoding cyclophilin from etiolated Vigna mungo seedlings

A full-length cDNA clone encoding cyclophilin gene of 848 bp, including a 519 bp open reading frame, has been isolated from the cDNA library constructed from etiolated seedlings of Vigna mungo (GenBank FN668732). The cDNA sequence showed 97% identity with Vigna radiata cyclophilin mRNA. The sequence was GC rich and lacked introns. The open reading frame encoded 172 amino acid polypeptide with molecular weight 18.3 kDa and theoretical pI 8.61. BlastP analysis indicated that its putative amino acid sequence shared 100% identity with several plant cyclophilins particularly legumes. The conserved seven amino acid residues region in V. mungo cyclophilin was RSGKPLH (present in legumes) instead of KSGKPLH, indicating its similarity to the cyclophilins of other legumes. This novel V. mungo cyclophilin gene will broaden the pool of plant cyclophilin genes for further studies.

Construction and Screening of Chickpea cDNA Library with Heterologous Legumin cDNA Probe

A cDNA library was constructed from developing chickpea cotyledons in the expression vector λ gt 11. The library when plated on X-gal and IPTG showed about 35% recombinants. These recombinants were screened using pea legumin cDNA probe. Of a total of 4 × 104 cDNA clones screened, 20 clones showed homology to legumin probe. The presence of cDNA insert in these clones was further confirmed by Southern blot hybridization.