Ajin D. Mandaokar

Insect-resistant transgenic brinjal plants

A synthetic cry1Ab gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis (Bt) was transferred to brinjal (eggplant) by cocultivating cotyledonary explants with Agrobacterium tumefaciens. Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and mRNA expression. Double-antibody sandwich ELISA analysis revealed Bt toxin protein expression in the transgenic plants. The expression resulted in a significant insecticidal activity of transgenic brinjal fruits against the larvae of fruit borer (Leucinodes orbonalis). The results also demonstrated that a synthetic gene based on monocot codon usage can be expressed in dicotyledonous plants for insect control.

Transgenic tomato plants resistant to fruit borer (Helicoverpa armigera Hubner)

Abstract A synthetic cry 1 Ac gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis ( Bt ) was transferred to tomato by cocultivating cotyledonary explants with Agrobacterium tumefaciens . Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and gene copy number in the transgenic plants. Double-antibody sandwich ELISA analysis revealed high levels of Bt ICP expression in the leaves of transgenic plants. The expression resulted in a high level of protection of transgenic plant leaves and fruits against the larvae of tomato fruit borer ( Helicoverpa armigera ). Limited field trial of the transgenic plants (T 1 generation) confirmed the high levels of insect protection.

Amino acid substitution in α‐helix 7 of Cry1Ac δ‐endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner

Insecticidal proteins or δ‐endotoxins of Bacillus thuringiensis are highly toxic to a wide range of agronomically important pests. The toxins are formed of three structural domains. The N‐terminal domain is a bundle of eight α‐helices and is implicated in pore formation in insect midgut epithelial membranes. All the δ‐endotoxins share a common hydrophobic motif of eight amino acids in α‐helix 7. A similar motif is also present in fragment B of diphtheria toxin (DT). Site‐directed mutagenesis of Cry1Ac δ‐endotoxin of B. thuringiensis was carried out to substitute its hydrophobic motif with that of DT fragment B. The mutant toxin was shown to be more toxic to the larvae of Helicoverpa armigera (cotton bollworm) than the wild‐type toxin. Voltage clamp analysis with planar lipid bilayers revealed that the mutant toxin opens larger ion channels and induces higher levels of conductance than the wild‐type toxin.

Bacillus thuringiensis cry1Ab gene confers resistance to potato against Helicoverpa armigera (Hubner).

SummaryHelicoverpa armigera is one of the important insect pests adversely affecting the yield of potatoes in India. A synthetic gene encoding the insecticidal crystal protein (Cry1Ab) ofBacillus thuringiensis (Bt) has been introduced into five genotypes of potato usingAgrobacterium tumefaciens. Southern analysis of DNA from transgenic plants confirmed the integration and copy number of the transgene. Double-antibody quantitative sandwich ELISA analysis demonstrated high levels of Cry1Ab protein expression in transgenic plants. Insect bioassays on the leaves of transgenic plants showed considerable protection against the larvae ofH. armigera in terms of leaf area consumed and larval weight reduction.

AFLP fingerprinting and genotypic characterization of some serovars of Bacillus thuringiensis.

Amplified fragment length polymorphism (AFLP)-based fingerprinting of 24 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 13 EcoR1 (+2) and Mse1 (+3) primer combinations for genotypic characterization. A high degree of polymorphism was established among the Bt serovars. A total of 1107 fragments ranging from 30–850 bp were generated out of which 1106 were polymorphic. Discrimination rates of different primer combinations at various band levels (1–5) among different Bt serovars were more than 90%. Cluster analysis revealed very low similarity values, ranging from 7–50%, among the Bt serovars indicating their remarkable genetic diversity. AFLP analysis establishes the molecular relatedness between the serovars and serotypes.

Cloning and Characterization of Legumin Storage Protein Gene of Chickpea (Cicer arietinum L)

Genomic DNA clones coding for legumin storage protein were isolated from chickpea (Cicer arietinum L.) subgenomic library using pea legumin cDNAs as a probe. The complete nucleotide sequence of the legumin gene (leg3) was determined. Sequence analysis indicated that leg3 contains 2022 bp long structural gene interrupted by three small introns. The gene encodes a polypeptide of 496 amino acids including a signal peptide of 21 amino acid residues and a-b subunit cleavage site. Leg3 polypeptide is rich in amide amino acids and contains 5 methionine and 6 cysteine amino acids. DNA sequence comparison showed about 70–80% identity with legumin genes from pea, soybean and broad bean. The 5’ flanking region contains regulatory elements such as TATA and legumin box and 3’ region contains a stop codon and two polyadenylation signals. Southern hybridisation indicated the presence of multiple copies of /eg3in chickpea and estimated about 10–12 copies per haploid genome. Northern blot hybridisation showed that leg3 expresses in developing seed and not in other tissues of chickpea indicating temporal and tissue-specific expression of this gene.

A fusion gene coding for two different δ-endotoxinsof Bacillus thuringiensis toxic to Plutella xylostella and usefulfor resistance management

Two truncated Bacillus thuringiensis δ-endotoxin genes, belonging to the classes cry1Ab and cry1B, and both coding for N-terminal toxic fragments of the corresponding crystal proteins, were translationally fused. Expression of the fusion gene driven by the cry1C promoter in Escherichia coli at a very high level resulted in a protein with enhanced toxicity to the diamondback moth (Plutella xylostella).

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.