Higher plant sucrose-phosphate synthase: Structure, function and regulation

523-529Sucrose-phosphate synthase (SPS) in higher plants catalyzes the penultimate and pivotal step of sucrose synthesis. SPS has a broad pH optimum for its activity and 'catalyzes a order bi bi reaction. SPS shows hyperbolic substrate saturation kinetics for both the substrates, UDP-glucose and fructose-6-phosphate. The enzyme is allosterically activated by glucose-6-phosphate and inhibited by Pi. These two effectors change the enzyme's affinity for both the substrates. SPS is believed to be a homodimer of 120-138 kd subunits with no interchain disulfide bridges. It has a conserved N-terminal portion with variable C-terminal part. Cloning of SPS gene has been accomplished from some plant species. SPS shows high sequence homology among the plant species. A glycine-rich region in the N-terminal portion is a distinctive feature of SPS from monocot plant species but is absent in dicots. Rice SPS gene has been mapped on chromosome 1. Genome organization study reveals that rice SPS gene consists of 12 exons and 11 introns. Light modulates SPS activity by covalent modification (coarse control) and allosteric regulation (fine control). In dark SPS is phosphorylated at a specific serine residue (Ser-158 in case of spinach SPS) by a specific protein kinase and becomes less active. Light activation of SPS activity involves dephosphorylation by a type 2A protein phosphatase. Covalent modification alters the kinetic properties of the enzyme. The diurnal and circadian rhythm of SPS activity is because of light regulation of de novo expression of SPS-phosphatase. The tissue metabolites, glucose-6-phosphate and Pi, not only alter the SPS activity but also affect the enzymes involved in covalent modulation of SPS. Glucose-6-phosphate inhibits SPS protein kinase and Pi inhibits SPS-phosphatase. The fluctuation of leaf Pi concentrations during light-dark transitions has been proposed to play a major role in the signal amplification of SPS regulation. The prospects of generation of transgenic plants overexpressing SPS have also been discussed

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Not AvailableRibonuclease P (RNase P), a ribonucleo-protein endoribonuclease, responsible for 5′ maturation of precursor tDNA, is well characterized in bacteria, yeast and human, but not in plant. Attempt has been made to partially purify and characterize nuclear RNase P from potato. cDNAs encoding two putative protein subunits of potato ribonuclease P (RNase P), StPop5 and StRpp25, were picked from potato EST library based on homology with respective human RNase P protein subunits. Both the cDNAs, 435 bp long StPop5 and 765 bp long StRpp25, were RT-PCR amplified, cloned and sequenced. StPop5 exhibited 46 % nucleotide sequence similarity to the hPop5 sequence. The deduced amino acid sequence of StPop5 had 23 % identity and 35 % similarity to hPop5. Both hRpp25 and StRpp25 had 46 % nucleotide sequence homology, and 17 % identity and 27 % similarity at a length of 271 amino acids. The molecular masses of purified 6× His-tagged recombinant StPop5 and StRpp25 proteins were 18 kDa and 33 kDa respectively. Potato nuclear RNase P was partially purified from leaves employing DEAE-Sephacel anion-exchange chromatography, and from floral buds employing DEAE-Sephacel and HiTrap Q anion-exchange chromatography, ammonium sulfate precipitation and gel filtration chromatography. Immunoprecipitation with polyclonal antisera, raised against recombinant StPop5 and StRpp25, demonstrated association of these two proteins with floral bud RNase P activity but not with leaf RNase P activity.Not Availabl

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Not AvailableBackground Development of chimeric Cry toxins by protein engineering of known and validated proteins is imperative for enhancing the efficacy and broadening the insecticidal spectrum of these genes. Expression of novel Cry proteins in food crops has however created apprehensions with respect to the safety aspects. To clarify this, premarket evaluation consisting of an array of analyses to evaluate the unintended effects is a prerequisite to provide safety assurance to the consumers. Additionally, series of bioinformatic tools as in silico aids are being used to evaluate the likely allergenic reaction of the proteins based on sequence and epitope similarity with known allergens. Results In the present study, chimeric Cry toxins developed through protein engineering were evaluated for allergenic potential using various in silico algorithms. Major emphasis was on the validation of allergenic potential on three aspects of paramount significance viz., sequence-based homology between allergenic proteins, validation of conformational epitopes towards identification of food allergens and physico-chemical properties of amino acids. Additionally, in vitro analysis pertaining to heat stability of two of the eight chimeric proteins and pepsin digestibility further demonstrated the non-allergenic potential of these chimeric toxins. Conclusions The study revealed for the first time an all-encompassing evaluation that the recombinant Cry proteins did not show any potential similarity with any known allergens with respect to the parameters generally considered for a protein to be designated as an allergen. These novel chimeric proteins hence can be considered safe to be introgressed into plants.Not Availabl

Gene pyramiding: A strategy for insect resistance management in <em>Bt</em> transgenic crops

283-291Preventing or delaying evolution of resistance in insects through adoption of judicial resistance management strategy is critical for the sustainable use of Bt transgenic crops. Strategies for resistance management in insect resistant Bt crops include: high dose strategy, moderate dose strategy, temporal- or time-specific toxins expression, refuge strategy, gene pyramiding/stacking, mixtures, rotations or mosaics of transgenic plants. Among these, pyramided/stacked plants have the potential to delay resistance more effectively than sequential deployment of single Bt toxin containing plants or in mosaics, even with relatively small and more economically acceptable refuge size. Resistance can be delayed for extended period even by reducing the refuge size if two or more toxin coding genes are pyramided in a single plant. If toxins are used independently, there is a possible risk of cross-resistance among the toxins

<span style="font-size: 21.5pt;mso-bidi-font-size:14.5pt;font-family:"Times New Roman","serif"; color:black">Characterization of genetic diversity of some serovars of <i>Bacillus thuringiensis </i><span style="font-size:21.0pt;mso-bidi-font-size:14.0pt; font-family:"Arial","sans-serif";color:black">by <span style="font-size: 22.0pt;mso-bidi-font-size:15.0pt;font-family:"Times New Roman","serif"; color:black;mso-bidi-font-weight:bold">RAPD </span></span></span>

897-901<span style="font-size: 15.5pt;mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif";="" color:black"="">RAPD based fingerprinting of 21 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 19 random decamer primers. A total of 172 polymorphic fragments, ranging in size from 161 -2789 bp, were amplified from 13 of the 19 primers. Pairwise genetic similarity analysis revealed very low similarity values, ranging from 3-68%, among the serovars of Bt, indicating high genetic divergence. Nineteen serovars of Bt fell in two major clusters and remaining two formed solitary clusters in the dendogram. Clustering of Bt strains established genetic relatedness between serovars and serotypes. It has been suggested that RAPD analysis can be used for genotypic characterization of Bt to complement flagellar serotyping. </span

Additional file 1: of Comprehensive in silico allergenicity assessment of novel protein engineered chimeric Cry proteins for safe deployment in crops

In silico assessment of pepsin digestibility sites in Cry1AcF and Cry1Aabc using Expasy peptide cutter. (XLSX 32 kb

Molecular Interaction-Based Exploration of the Broad Spectrum Efficacy of a Bacillus thuringiensis Insecticidal Chimeric Protein, Cry1AcF

Bacillus thuringiensis insecticidal proteins (Bt ICPs) are reliable and valuable options for pest management in crops. Protein engineering of Bt ICPs is a competitive alternative for resistance management in insects. The primary focus of the study was to reiterate the translational utility of a protein-engineered chimeric Cry toxin, Cry1AcF, for its broad spectrum insecticidal efficacy using molecular modeling and docking studies. In-depth bioinformatic analysis was undertaken for structure prediction of the Cry toxin as the ligand and aminopeptidase1 receptors (APN1) from Helicoverpa armigera (HaAPN1) and Spodoptera litura (SlAPN1) as receptors, followed by interaction studies using protein-protein docking tools. The study revealed feasible interactions between the toxin and the two receptors through H-bonding and hydrophobic interactions. Further, molecular dynamics simulations substantiated the stability of the interactions, proving the broad spectrum efficacy of Cry1AcF in controlling H. armigera and S. litura. These findings justify the utility of protein-engineered toxins in pest management

Small but mighty RNA-mediated interference in plants

7-24RNA silencing is a conserved phenomenon of regulation of gene expression by small RNAs derived from cleavage of double-stranded RNA (dsRNA). The present review deals with three overlapping modes of small RNA-mediated silencing particularly in plants. In case of post-transcriptional gene silencing (PTGS), Dicer, an endonuclease, cleaves dsRNA to produce-21nt-long small interfering RNAs (siRNAs), which guide RISC, another nuclease complex, to destroy specific target mRNAs based on sequence complementarity with the siRNA. Another class of siRNAs of 25nt-long is also produced from dsRNA by Dicer, different from that generates 21nt-long siRNA. These longer siRNAs are probably involved in systemic silencing during PTGS and guide methylation of both DNA and histone, and induce heterochromatinization and consequent transcriptional repression of the targeted gene. Both siRNA-mediated PTGS and epigenetic modification of the genome are considered as defense mechanisms to protect against invading viruses, transposons or aberrantly expressing transgenes. Regulation of expression of endogenous genes is mediated by another class of 21nt-long small RNAs called microRNAs (miRNA). Genes encoding the miRNAs are present either in the intergenic regions, introns or coding regions of the plant genome. Cleavage of a stem-loop precursor transcript called pre-miRNA, by another class of Dicer generates miRNAs, which in association with nuclease complex similar to RISC, if not identical, either degrade target mRNA or cause translational repression. The applications of RNA silencing in functional genomics and crop improvement are discussed

Rapid and efficient <em>Agrobacterium </em> mediated transformation of early scutellum derived calli of <em>indica</em> rice

20-28Rice is a staple food for humans and its demand in 2035 has been put at 852 million tons. Knowledge on genes and genome architecture helps in better understanding of growth and development mechanisms for crop improvement. Transgenic crops may offer a solution by means of higher yield and resistance to biotic and abiotic stresses. In this context, modification of Agrobacterium mediated transformation protocol for indica rice cultivar is imperative to increase transformation efficiency and reduce duration of transgenic development. Here, we developed an efficient Agrobacterium mediated transformation protocol using early scutellum derived calli of the indica rice cultivar Pusa Sugandh 2. Competency of 3, 4, 5 and 6 day old primary calli was compared with 21- day old secondary calli for Agrobacterium mediated transformation using a modified pCAMBIA 1304 harbouring GFP-GUS fusion gene driven by maize ubiquitin 1 promoter. The highest competency with stable transformation efficiency of 51% was observed for 5-6 day old primary calli. Molecular analysis confirmed stable integration of the transgene. Transgenic lines of Pusa Sugandh 2 were developed within a short period of two months using 5-6 day old primary calli

Brassica juncea Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines

Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines