Sugey Ramona Sinagawa-García

Accumulation, assembly, and digestibility of amarantin expressed in transgenic tropical maize.

An amaranth (Amaranthus hypochondriacus) 11S globulin cDNA, encoding one of the most important storage proteins (amarantin) of the seed, with a high content of essential amino acids, was used in the transformation of CIMMYT tropical maize genotype. Constructs contained the amarantin cDNA under the control of a tissue-specific promoter from rice glutelin-1 (osGT1) or a constitutive (CaMV 35S) promoter with and without the first maize alcohol dehydrogenase intron (AdH). Southern-blot analysis confirmed the integration of the amarantin cDNA, and copy number ranged from one to more than ten copies per maize genome. Western-blot and ultracentrifugation analyses of transgenic maize indicate that the expressed recombinant amarantin precursors were processed into the mature form, and accumulated stably in maize endosperm. Total protein and some essential amino acids of the best expressing maize augmented 32% and 8–44%, respectively, compared to non-transformed samples. The soluble expressed proteins were susceptible to digestion by simulated gastric and intestinal fluids, and it is suggested that they show no allergenic activity. These findings demonstrate the feasibility of using genetic engineering to improve the amino acid composition of grain crops.

Visual spectinomycin resistance ( aadA au ) gene for facile identification of transplastomic sectors in tobacco leaves

Identification of a genetically stable Nicotiana tabacum (tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea bar (barau) gene that confers a golden leaf phenotype and a spectinomycin resistance (aadA) gene that is necessary for the introduction of the barau gene in the plastid genome. We developed a novel aadA gene that fulfills both functions: it is a conventional selectable aadA gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the clpP1 plastid gene by aurea aadAau transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new aadAau genes are useful to query rare events in large populations and for in planta manipulation of the plastid genome.

Study of Plastid Genome Stability in Tobacco Reveals That the Loss of Marker Genes Is More Likely by Gene Conversion Than by Recombination between 34-bp loxP Repeats

In transformed tobacco (Nicotiana tabacum) plastids, we flank the marker genes with recombinase target sites to facilitate their posttransformation excision. The P1 phage loxP sites are identical 34-bp direct repeats, whereas the phiC31 phage attB/attP sites are 54- and 215-bp sequences with partial homology within the 54-bp region. Deletions in the plastid genome are known to occur by recombination between directly repeated sequences. Our objective was to test whether or not the marker genes may be lost by homologous recombination via the directly repeated target sites in the absence of site-specific recombinases. The sequence between the target sites was the barau gene that causes a golden-yellow (aurea) leaf color, so that the loss of the barau gene can be readily detected by the appearance of green sectors. We report here that transplastomes carrying the barau gene marker between recombinase target sites are relatively stable because no green sectors were detected in approximately 36,000 seedlings (Nt-pSS33 lines) carrying attB/attP-flanked barau gene and in approximately 38,000 seedlings (Nt-pSS42 lines) carrying loxP-flanked barau gene. Exceptions were six uniformly green plants in the Nt-pSS42-7A progeny. Sequencing the region of plastid DNA that may derive from the vector indicated that the barau gene in the six green plants was lost by gene conversion using wild-type plastid DNA as template rather than by deletion via directly repeated loxP sites. Thus, the recombinase target sites incorporated in the plastid genome for marker gene excisions are too short to mediate the loss of marker genes by homologous recombination at a measurable frequency.

High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly.

Evaluation of Biochemical Components from Pterophylla beltrani (Bolivar & Bolivar) (Orthoptera: Tettigoniidae): A Forest Pest from Northeastern Mexico

Abstract. Insects are a biotic resource used for human medicine, nutrition, and industry. Many are a potential source of compounds such as proteins, peptides, secondary metabolites, and polymers like silk, chitin, and chitosan for industry and biotechnology. The Mexican katydid, Pterophylla beltrani Bolivar & Bolivar, is an important insect pest that defoliates oaks (Quercus spp.) and other trees where it feeds and becomes abundant during summer in forests in Northeast Mexico. Several strategies based on extracts of compounds and sustainable metabolites for use as raw material have been proposed for its control and use. Antioxidant, chitin, and chitosan content of P. beltrani were analyzed. Extracted aqueous residue was evaluated for effect on fungal growth, and chitosan was evaluated to supplement the growth of entomopathogenic fungi. Aqueous extract from P. beltrani flour contained phenolic compounds (1.08 ± 0.049 GAE mg g-1) that had little anti-fungal effect against three species of fungi, but did inhibit the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) Sorokin. Yield of chitin was 11.8% and produced 58.8% chitosan, indicating the insect is a potential source of biochemical compounds. This could help support sustainable management of this pest.

Entomochemicals from Pterophylla beltrani Bolivar and Bolivar: Antioxidants and Other Metabolites

Abstract. Insects are sources of diverse natural metabolites or entomochemicals with different properties. Extraction of entomochemicals depends on the polarity of solvent and affinity of metabolites, important for yield of bioactive entomochemicals. Antioxidants and entomochemicals of Pterophylla beltrani Bolivar and Bolivar (Orthoptera: Tettigoniidae) have been studied. In this work, several solvents were used and compared in relation to their capacity to extract entomochemicals from P. beltrani flour. Differences in yield of phenolics and antioxidant capacity between solvents were observed using methods based on Folin-Ciocalteu reagent, DPPH, and ABTS, respectively. Water was the best solvent to extract phenolics and antioxidant compounds from P. beltrani; while propanol was in general the solvent that resulted in least entomochemicals. Water extraction yielded 187 GAE mg/g flour for total phenolic compounds, 121.75 mM TE/g flour with the DPPH assay, and 362.6 mM TE/g flour with the ABTS assay, while propanol extraction yielded 0.74 GAE mg, 47.25 mM TE, and 55.45 mM TE per gram of flour for total phenolic compounds and antioxidants with DPPH and ABTS assays, respectively. Methanol and ethanol extractions yielded intermediate values. Alkaloids and sterols were detected using colorimetric assays of some extracts. Twenty compounds of alkaloids, phenolic compounds, amino acid derivatives, tryptamine derivatives, pyrazine derivatives, organic acids, fatty amides, and terpenes were identified using mass spectrometry; 20 unknown compounds also were detected. This makes P. beltrani a potential source of diverse entomochemicals. Further chemical characterization is needed to understand the entomochemicals and their roles in physiology and ecological interactions of P. beltrani.

Digestive Proteinases and Antioxidant Capacity from Pterophylla beltrani Bolivar & Bolivar1 Fed Two Natural Diets

Abstract. Insect diets are complex and usually contain antinutritional factors; because of this, modification of the digestive process and strategies to cope with the factors determines adaptation of each species. Pterophylla beltrani Bolivar & Bolivar is a forest pest that has two feeding regimes; P. beltrani feeds first on diverse vegetation but changes during the second regime to consume foliage of oaks (Quercus spp.). The goal of this work was to assess the status of digestive proteinases and phenolic-antioxidants in the two feeding regimes, as indicators of changes related to diet variation. Trypsin-like activity was confirmed by hydrolysis of a synthetic chromogenic substrate. Units of proteolytic activity were 25.2 to 46.1 for the first regime and 22.6 to 40.2 for the second. No differences in proteolytic profiles were detected between regimes. Total phenolic content was 1,152.2 and 759 GAE/ml for the first regime and 832.2 and 552.2 GAE/ml for the second when samples were extracted in water or ethanol, respectively. Differences in the antioxidant capacity also were observed in individuals from the different feeding regimes — large amounts of phenolics for the first regime and greater antioxidant capacity for the second. Diet allows differential accumulation of antioxidants that might be associated with physiological and ecological implications yet to be discovered.