Vudem Dashavantha Reddy

Molecular characterization, physicochemical properties, known and potential applications of phytases: An overview

Phytases (myo-inositol hexakisphosphate phosphohydrolases) hydrolyze the phosphate ester bonds of phytate-releasing phosphate and lower myo-inositol phosphates and/or myo-inositol. Phytases, in general, are known to enhance phosphate and mineral uptake in monogastric animals such as poultry, swine, and fish, which cannot metabolize phytate besides reducing environmental pollution significantly. In this study, the molecular, biophysical, and biochemical properties of phytases are reviewed in detail. Alterations in the molecular and catalytic properties of phytases, upon expression in heterologous hosts, are discussed. Diverse applications of phytases as feed additives, as soil amendment, in aquaculture, development of transgenic organisms, and as nutraceuticals in the human diet also are dealt with. Furthermore, phytases are envisaged to serve as potential enzymes that can produce versatile lower myo-inositol phosphates of pharmaceutical importance. Development of phytases with improved attributes is an important area being explored through genetic and protein engineering approaches, as no known phytase can fulfill all the properties of an ideal feed additive.

Transgenic rice plants expressing the snowdrop lectin gene (gna) exhibit high-level resistance to the whitebacked planthopper (Sogatella furcifera)

Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T1 to T5 generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper .

Expression of pigeonpea hybrid‐proline‐rich protein encoding gene (CcHyPRP) in yeast and Arabidopsis affords multiple abiotic stress tolerance

A hybrid-proline-rich protein encoding gene (CcHyPRP) has been isolated and characterized, for the first time, from the subtracted cDNA library of pigeonpea (Cajanus cajan L.) plants subjected to drought stress. Functionality of CcHyPRP has been validated for abiotic stress tolerance using the heterologous yeast and Arabidopsis systems. The CcHyPRP contained a repetitive proline-rich (PR) N-terminal domain and a conserved eight cysteine motif (8CM) at the C-terminus. Southern analysis disclosed single-copy nature of CcHyPRP gene in the pigeonpea genome. Northern analysis revealed higher levels of CcHyPRP transcripts in PEG, NaCl, heat (42 degrees C), cold and ABA-treated plants compared with the weak signals observed in the untreated plants, suggesting stress-responsive nature of the CcHyPRP gene. In yeast, expression of CcHyPRP imparted marked tolerance against abiotic stresses exerted by PEG, high temperature, NaCl and LiCl. Transgenic Arabidopsis lines, expressing CcHyPRP under the control of CaMV35S and rd29A promoters, when subjected to PEG, mannitol, NaCl, LiCl and heat (42 degrees C) stress, developed into healthy plants with profuse root system and increased biomass in contrast to the weak-stunted wild-type plants. The CcHyPRP-transgenics driven by stress-inducible rd29A exhibited similar stress-tolerance as that of CaMV35S-lines without any negative effects on plant morphology, implying that stress-inducible promoters are preferable for production of stress tolerant transgenics. The overall results amply demonstrate the profound effect of CcHyPRP in bestowing multiple abiotic stress tolerance at cellular and whole plant levels. Accordingly, the multipotent CcHyPRP seems promising as a prime candidate gene to fortify crop plants with abiotic stress tolerance.

Isolation and characterization of a pigeonpea cyclophilin (CcCYP) gene, and its over‐expression in Arabidopsis confers multiple abiotic stress tolerance

A full-length cDNA clone of pigeonpea (Cajanus cajan L.) encoding cyclophilin (CcCYP) has been isolated from the cDNA library of plants subjected to drought stress. Amino acid sequence of CcCYP disclosed similarity with that of single-domain cytosolic cyclophilins of various organisms. Expression profile of CcCYP in pigeonpea plants is strongly induced by different abiotic stresses, indicating its stress-responsive nature. Compared to the control plants, the transgenic Arabidopsis lines expressing CcCYP exhibited high-level tolerance against major abiotic stresses, viz., drought, salinity and extreme temperatures as evidenced by increased plant survival, biomass, chlorophyll content and profuse root growth. The CcCYP transgenics, compared to the controls, revealed enhanced peptidyl-propyl cis-trans isomerase (PPIase) activity under stressed conditions, owing to transcriptional activation of stress-related genes besides intrinsic chaperonic activity of the cyclophilin. The transgenic plants subjected to salt stress exhibited higher Na(+) ion accumulation in roots as compared to shoots, while a reverse trend was observed in the salt-stressed control plants, implicating the involvement of CcCYP in the maintenance of ion homeostasis. Expression pattern of CcCYP:GFP fusion protein confirmed the localization of CcCYP predominantly in the nucleus as revealed by intense green fluorescence. The overall results amply demonstrate the implicit role of CcCYP in conferring multiple abiotic stress tolerance at whole-plant level.

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T0, 2T0, and 3T0 showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T1 progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T1 plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T1 plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T2 progenies of 1T0 conformed to dihybrid segregation of 15 resistant:1 susceptible plants.

Characterization of expressed sequence tags (ESTs) of pigeonpea (Cajanus cajan L.) and functional validation of selected genes for abiotic stress tolerance in Arabidopsis thaliana.

Pigeonpea, a major grain legume crop with remarkable drought tolerance traits, has been used for the isolation of stress-responsive genes. Herein, we report generation of ESTs, transcript profiles of selected genes and validation of candidate genes obtained from the subtracted cDNA libraries of pigeonpea plants subjected to PEG/water-deficit stress conditions. Cluster analysis of 124 selected ESTs yielded 75 high-quality ESTs. Homology searches disclosed that 55 ESTs share significant similarity with the known/putative proteins or ESTs available in the databases. These ESTs were characterized and genes relevant to the specific physiological processes were identified. Of the 75 ESTs obtained from the cDNA libraries of drought-stressed plants, 20 ESTs proved to be unique to the pigeonpea. These sequences are envisaged to serve as a potential source of stress-inducible genes of the drought stress-response transcriptome, and hence may be used for deciphering the mechanism of drought tolerance of the pigeonpea. Expression profiles of selected genes revealed increased levels of m-RNA transcripts in pigeonpea plants subjected to different abiotic stresses. Transgenic Arabidopsis lines, expressing Cajanus cajan hybrid-proline-rich protein (CcHyPRP), C. cajan cyclophilin (CcCYP) and C. cajan cold and drought regulatory (CcCDR) genes, exhibited marked tolerance, increased plant biomass and enhanced photosynthetic rates under PEG/NaCl/cold/heat stress conditions. This study represents the first report dealing with the isolation of drought-specific ESTs, transcriptome analysis and functional validation of drought-responsive genes of the pigeonpea. These genes, as such, hold promise for engineering crop plants bestowed with tolerance to major abiotic stresses.

Structural organization and classification of cytochrome P450 genes in flax (Linum usitatissimum L.)

Flax CYPome analysis resulted in the identification of 334 putative cytochrome P450 (CYP450) genes in the cultivated flax genome. Classification of flax CYP450 genes based on the sequence similarity with Arabidopsis orthologs and CYP450 nomenclature, revealed 10 clans representing 44 families and 98 subfamilies. CYP80, CYP83, CYP92, CYP702, CYP705, CYP708, CYP728, CYP729, CYP733 and CYP736 families are absent in the flax genome. The subfamily members exhibited conserved sequences, length of exons and phasing of introns. Similarity search of the genomic resources of wild flax species Linum bienne with CYP450 coding sequences of the cultivated flax, revealed the presence of 127 CYP450 gene orthologs, indicating amplification of novel CYP450 genes in the cultivated flax. Seven families CYP73, 74, 75, 76, 77, 84 and 709, coding for enzymes associated with phenylpropanoid/fatty acid metabolism, showed extensive gene amplification in the flax. About 59% of the flax CYP450 genes were present in the EST libraries.

Pyramided rice lines harbouring Allium sativum (asal) and Galanthus nivalis (gna) lectin genes impart enhanced resistance against major sap- sucking pests

We have developed transgene pyramided rice lines, endowed with enhanced resistance to major sap-sucking insects, through sexual crosses made between two stable transgenic rice lines containing Allium sativum (asal) and Galanthus nivalis (gna) lectin genes. Presence and expression of asal and gna genes in pyramided lines were confirmed by PCR and western blot analyses. Segregation analysis of F₂ progenies disclosed digenic (9:3:3:1) inheritance of the transgenes. Homozygous F₃ plants carrying asal and gna genes were identified employing genetic and molecular methods besides insect bioassays. Pyramided lines, infested with brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH), proved more effective in reducing insect survival, fecundity, feeding ability besides delayed development of insects as compared to the parental transgenics. Under infested conditions, pyramided lines were found superior to the parental transgenics in their seed yield potential. This study represents first report on pyramiding of two lectin genes into rice exhibiting enhanced resistance against major sucking pests. The pyramided lines appear promising and might serve as a novel genetic resource in rice breeding aimed at durable and broad based resistance against hoppers.

Structural and kinetic properties of Bacillus subtilis S-adenosylmethionine synthetase expressed in Escherichia coli

S-adenosylmethionine (SAM) synthetase (EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionine using l-methionine and ATP as substrates. SAM synthetase gene (metE) from Bacillus subtilis was cloned and over-expressed, for the first time, in the heterologus host Escherichia coli as an active enzyme. Size-exclusion chromatography (SEC) revealed a molecular weight of ~180 kDa, suggesting that the enzyme is a homotetramer stabilized by non-covalent interactions. SAM synthetase exhibited optimal activity at pH 8.0 and 45 degrees C with the requirement of divalent cation Mg(2+), and stimulated by the monovalent cation K(+). The enzyme followed sequential mechanism with a V(max) of 0.362 micromol/min/mg, and a K(m) of 920 microM and 260 microM for ATP and l-methionine, respectively. The urea-induced unfolding equilibrium of the recombinant enzyme revealed a multistate process, comprising partially unfolded tetramer, structural dimer, structural monomer and completely unfolded monomer, as evidenced by intrinsic and extrinsic fluorescence, circular dichroism (CD) and SEC. Absence of trimer in the SEC implicates that the enzyme is a dimer of dimer. Concordance between results of SEC and enzyme activity in the presence of urea amply establishes that tetramer alone with intersubunit active site(s) exhibits enzyme activity.

Current status of genetic engineering in cotton (Gossypium hirsutum L): an assessment.

Abstract Cotton is considered as the foremost commercially important fiber crop and is deemed as the backbone of the textile industry. The productivity of cotton crop, worldwide, is severely hampered by the occurrence of pests, weeds, pathogens apart from various environmental factors. Several beneficial agronomic traits, viz., early maturity, improved fiber quality, heat tolerance, etc. have been successfully incorporated into cotton varieties employing conventional hybridization and mutation breeding. Crop losses, due to biotic factors, are substantial and may be reduced through certain crop protection strategies. In recent years, pioneering success has been achieved through the adoption of modern biotechnological approaches. Genetically engineered cotton varieties, expressing Bacillus thuringiensis cry genes, proved to be highly successful in controlling the bollworm complex. Various other candidate genes responsible for resistance to insect pests and pathogens, tolerance to major abiotic stress factors such as temperature, drought and salinity, have been introduced into cotton via genetic engineering methods to enhance the agronomic performance of cotton cultivars. Furthermore, genes for improving the seed oil quality and fiber characteristics have been identified and introduced into cotton cultivars. This review provides a brief overview of the various advancements made in cotton through genetic engineering approaches.