Molecular cloning, characterization and expression of lipoxygenase 2 (lox-2) isozyme from Indian soybean [Glycine max (L.) Merrill] cv. Pusa 16.

The consumption of soybean is limited worldwide, despite being highly nutritious and having versatile uses due to the presence of grassy, beany and rancid off-flavour. The lipoxygenase-2 (LOX-2) is the key enzyme responsible for the production of volatiles released from the beans, which cause off-flavour in soy products. In this study, a 2.6-kb full-length lox2 gene (NCBI accession No. JQ929619.1) was isolated and cloned from soybean (Glycine max L. Merril) cv. Pusa 16. The cloned cDNA sequence of lox2 gene showed the complete open reading frame (ORF) of a putative protein, having 866 amino acids with start codon present at the foremost position and stop codon at the end. The theoretical pI of predicted protein was 6.22. A hydropathy profile calculated from the amino acid sequence resembled those of dicot LOXs, suggesting conservation of the secondary structure of these enzymes. The LOX-2 showed conserved six Histidine residues within a span of 520 to 590 amino acid position, a signature element for the enzyme activity. The lox2 gene was expressed using pET vector in prokaryotic expression system. The recombinant LOX-2 protein was purified after induction with IPTG (isopentyl thiogalactoside). A prominent band of 97 kDa was observed, when affinity purified fractions were analyzed by SDS-PAGE. The purified protein was characterized for the enzyme activity, substrate preference and K(m). Inhibitor studies with natural antioxidant molecules present in soybean revealed alpha-tocopherol to be the most effective inhibitor of LOX-2.

Regulation of hordein synthesis in barley high lysine mutant Notch-2.

Genomic DNA isolated from barley cv. NP 113 and its high lysine mutant Notch-2, and restricted with different restriction enzymes was hybridized with B1 and C-hordein DNA probes. Similar Southern hybridization patterns were observed between NP 113 and Notch-2. Dot blot hybridization analysis of RNA isolated at different developmental stages and from different tissues of seed showed temporal as well as tissue specific expression. The results obtained indicate that regulation at the level of transcription/post transcription may be responsible for lower accumulation of hordein in mutant Notch-2.

Effect of water stress on proline content and transcript levels in Lathyrus sativus.

Response of Lathyrus sativus plants to water stress showed that ABA responsive genes such as PLE 25, TAS 14 and RAB 17 are synthesized constitutively, the levels of which decline gradually with increase in water stress or ABA levels. Proline accumulation was highest in leaves (65-fold) followed by stem (56-fold), root (38-fold) and marginal increase in etiolated seedlings. Proline increase was also observed in plant parts not exposed to light.

In vitro kinetics of soybean lipoxygenase with combinatorial fatty substrates and its functional significance in off flavour development.

Lipoxygenase (Lox) mediated oxidation of polyunsaturated fatty acids (PUFA) in mature soya seeds results in objectionable flavour. In the present study, Lox isozymes were purified to near homogeneity (107-fold). Lox-2 and 3 displayed remarkable kinetic preference (1.7 and 1.5-fold, respectively) for low PUFA ratios (LA/LeA) (PRs) among the selected PUFA combinations. Lox-1 displayed no specific preference. Pure Lox-1 displayed unbiased response towards substrates with marginal preference (1.2-fold) for linoleic acid at its optimum pH. Volatile compounds profiling showed a direct relationship between PRs and hexanal to trans-2-hexenal (1.47, 2.24 and 18.90 for 2, 7 and 15 PRs, respectively) ratio. The off-flavour determining parameters like TBA value, carbonyl value and lipid hydroperoxides (LHPODs) exhibited significant negative correlation (0.76, 0.74, 0.72; p<0.0001) in selected soya genotypes displaying varied PRs and significant positive correlation (0.89, 0.81. 0.89; p<0.0001) with ratio of PI (polyene index) to PRs - suggesting the plausible significance of PUFA ratios in biological lipid peroxidation.

In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae.

Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5 percentage yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80 degree C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera.

Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.

Amino acid compositions of different protein fractions in developing grains of NP 113 barley and its high lysine Notch-2 mutant.

The percent distributions of protein fractions namely albumin + globulin, prolamine and glutelin were studied in developing grains of NP 113 barley and its high lysine mutant Notch-2. During development the percentage of albumin + globulin fraction decreased in NP 113, while those of prolamine and glutelin remained unchanged. The increase in prolamine was substantial from 24 to 31DAA. In Notch-2 the trend followed by albumin + globulin and prolamine was like that in NP 113, while the glutelin fraction showed an increase as compared to 10 DAA. The percent of albumin + globulin was slightly higher in Notch-2 as compared to NP 113. The absolute amount (mg/grain) of all the protein fractions increased during development in both NP 113 and its mutant Notch-2. During the grain development the prolamine content was substantially lower in the mutant than in the parent NP 113. The albumin + globulin content per endosperm was in general also higher in NP 113 than Notch-2. Amino acid analysis of the protein fractions did not reveal significant changes in lysine between NP 113 and Notch-2. Thus, the improvement in lysine in the mutant is primarily due to reduced synthesis of the prolamine fraction and not due to an increase in lysine in the mutant hordein fraction. Part of the improvement in lysine may also be due to increase in the percentage of albumin + globulin fractions which is lysine rich.

Cloning of acyl-acyl carrier protein (ACP) thioesterase gene from Brassica juncea.

Acyl-acyl carrier protein (ACP) thioesterase is the chain-length-determining enzyme in de novo biosynthesis of plant fatty acids. For cloning the gene encoding acyl-ACP thioesterase from Brassica juncea, genomic DNA was used as a template to amplify a 0.7 kb thioesterase fragment in a PCR with the primers designed from the known sequences available in the GenBank. This 0.7 kb fragment was used as a probe to study the expression of the gene in developing seeds and also to screen a genomic library of B. juncea constructed in lambdaEMBL-3 to get the full length of the gene. A 4.0 kb BamHI fragment containing the full gene was finally cloned in a plasmid vector from a recombinant phage clone lambda5.12 after a series of screening, sub-cloning and Southern hybridization.

Hybridization and selection for improving seed protein in barley.

Hybridization followed by continuous selection of lines of barley from different cross-combinations involving high protein-high lysine genotypes and the agronomically superior strains resulted in breaking the negative correlations between 1000 grain weight and high protein content and high DBC values. The methodology of DBC-Kjeldahl protein adopted in the present study is likely to be useful in identifying high lysine lines. The present study has shown considerable variability with respect to protein content and grain weight and has provided interesting genotypes which can be used in synthesising lines with improved nutritional quality and productivity in barley. The success in breaking the undesirable linkages to factors that impair the endosperm development is due to sufficient genetic variability in the initial breeding material as well as the use of suitable breeding procedures like the full-sib mating in the early segregating generations.