Identification of glycinin and beta-conglycinin subunits that contribute to the increased protein content of high-protein soybean lines

Seed protein concentration of commercial soybean cultivars calculated on a dry weight basis ranges from approximately 37 to 42% depending on genotype and location. A concerted research effort is ongoing to further increase protein concentration. Several soybean plant introductions (PI) are known to contain greater than 50% protein. These PIs are exploited by breeders to incorporate the high-protein trait into commercial North American cultivars. Currently, limited information is available on the biochemical and genetic mechanisms that regulate high-proteins. In this study, we have carried out proteomic and molecular analysis of seed proteins of LG00-13260 and its parental high-protein lines PI 427138 and BARC-6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that the high-protein lines accumulated increased amounts of beta-conglycinin and glycinins, when compared with Williams 82. High-resolution two-dimensional electrophoresis utilizing pH 4-7 and pH 6-11 ampholytes enabled improved resolution of soybean seed proteins. A total of 38 protein spots, representing the different subunits of beta-conglycinin and glycinin, were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. High-protein was correlated with an increase in the accumulation of most of the subunits representing beta-conglycinin and glycinin. Comparisons of the amino acid profiles of high-protein soybean lines revealed that the concentration of sulfur amino acids, a reflection of protein quality, was not influenced by the protein concentration. Southern blot analysis showed the presence of genotypic variation at the DNA level between PI 427138 and BARC-6 for the genes encoding group1 glycinin, beta-conglycinin, Bowman-Birk inhibitor (BBI), and the Kunitz trypsin inhibitor (KTI). LG00-13260 inherited the allelic variants of the parental line PI 427138 for glycinin, beta-conglycinin, and KTI, while BBI was inherited from the parental line BARC-6. The results of our study indicate that high-seed protein concentration is attributed to greater accumulation of specific components of beta-conglycinin and glycinin subunits presumably mediated by preferential expression of these genes during seed development.     

Role of outer membrane proteins in immunity against murine salmonellosis--1. Antibody response to crude outer membrane proteins of Salmonella typhimurium

The humoral immune response to crude outer membrane proteins (comp) of S. typhimurium in mice has been characterized. Maximal and quicker antibody response was observed when 50 micrograms of comp was injected intraperitoneally. The comp of smooth C5 strain of S. typhimurium evoked antibody response to both lipopolysaccharide (LPS) and proteins. Absorption of these sera with LPS coated erythrocytes eliminated the antibodies to LPS completely, while the antibody level to protein was left unaltered. The comp from rough mutant (lacking O-specific chain of LPS) of S. typhimurium elicited antibodies to proteins but not to LPS. These results indicate the concomitant production of antibodies to Salmonella outer membrane proteins also. The significance of such antibodies in protection and diagnosis has been discussed.     

Mutations: Incidence in Drosophila melanogaster Reared on Irradiated Medium

An increase in the rate of mutation has been found in Drosophila melanogaster reared on a basic medium that was irradiated with a sterilizing dose (150,000 rads) of cobalt-60 gamma rays. In Muller-5 tests, sex-linked recessive lethals occurred only in the F(2) progenies of the male test flies obtained from breeding the parent flies on irradiated medium, while visible changes occurred in experimental cultures from both the control and irradiated media. The frequency of sex-linked recessive lethals was 0.35, 0.55, and 0.8 percent in three independent experiments. Visible changes were two to six times more frequent in the irradiated series than in the controls.     

Development and standardization of a piezo electric immunobiosensor for foot and mouth disease virus typing

An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot and mouth disease (FMD) diagnosis and virus typing. A 6MHz quartz crystal was used as the frequency determining element. Foot and mouth disease virus (FMDV) type specific antibody raised in rabbits/monoclonal antibody was coated on the crystal surface and the resonance measured. One microlitre of the 10% aqueous suspension of the clinical sample (tongue or foot epithelium) was applied on both surfaces of the crystal and the resonance recorded. A difference in resonance of more than -2.5Hz was obtained in positive samples (homologous antigen and antibody). The test was standardized initially using various dilutions of FMD tissue culture antigen. Repeatability and sensitivity were also tested and it was found that the crystals could be washed and reused eight times. The test could be used for FMDV type specifically and no cross-reaction between FMDV types was observed. The shelf-life of the antibody-coated crystal stored at room temperature was 18 weeks. Application of the biosensor test to the FMDV clinical samples confirmed virus typing results when compared with enzyme-linked immunoabsorbent assay (ELISA) and it could also detect virus in ELISA negative samples and mixed virus infections.     

Early antibody responses of cattle for foot-and-mouth disease quadrivalent double oil emulsion vaccine

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals. The multiplicity of FMDV serotypes in animals poses a central problem in the policy of vaccination and is of much concern to health authorities. Hence it is the practice of vaccination with polyvalent vaccine for prophylactic measure. In the present report, we analysed the early antibody responses elicited by FMDV quadrivalent (FMDV O, A, C and Asia 1 serotypes) double emulsion (Montanide ISA 206) vaccines in cattle. We observed variations between various viral serotypes in eliciting early antibody response although neutralizing antibody response against all the four serotypes were detected as early as fourth day following vaccination. The duration of immunity also appeared to maintain for long period. The neutralizing antibody titres were maintained well above 2log(10) even after 6 months of vaccination irrespective of serotypes. Thus, allows the possibilities of two vaccinations per year for the maintenance of herd immunity.     

Effect of probiotic supplementation on serum/yolk cholesterol and on egg shell thickness in layers

1. The effect of probiotic supplementation on egg production, on serum and yolk cholesterol and on egg shell thickness in 24 White Leghorn layers was studied from 28–38 weeks of age.

2. In 3 treatments the diet was supplemented with 0, 100 and 150 mg probiotic/kg food.

3. In the 100 mg probiotic group, egg production improved by 5%, and shell thickness improved slightly, with fewer thin‐shelled eggs than in the control (8.6% compared to 18.6%).

4. The initial serum cholesterol concentration of 170.2 mg/dl in control birds remained similar throughout the 10‐week experimental period, whereas in the 150 mg group the initial value of 176.5 mg/dl decreased to 114.3 mg by week 10.

5. Yolk cholesterol concentration was 14.69 mg in the control group and 11.28 and 11.37 mg/g in the 100 and 150 mg probiotic groups respectively. Overall mean total egg cholesterol was thus reduced by probiotic supplementation.     

Mycorrhizal (Rhizophagus Intraradices) Symbiosis and Fe and Zn Availability in Calcareous Soil

Greenhouse experiment was conducted to assess the iron (Fe) and zinc (Zn) fractionation patterns in soils of arbuscular mycorrhizal (AM) fungus-inoculated and uninoculated maize plants fertilized with varying levels of Fe and Zn. Soil samples were collected for Fe and Zn fractions and available Fe, Zn and phosphorus (P) contents besides organic and biomass carbon (BMC), soil enzymes and glomalin. Major portion of Fe and Zn fractionations was found to occur in the residual form. Mycorrhizal symbiosis increased the organically bound forms of Fe and Zn while reducing the crystalline oxide, residual Fe and Zn fractions, indicating the transformation of unavailable forms into available forms. Soil enzymes, viz. dehydrogenase and acid phosphatase activities in M+ soils, were significantly higher than M? soil consistently. Overall, the data suggest that mycorrhizal symbiosis enhanced the availability of Fe and Zn as a result of preferential fractionation and biochemical changes that may alleviate micronutrient deficiencies in calcareous soil.

Abbreviations: AM: arbuscular mycorrhiza; Fe: Iron; Zn: Zinc; P: Phosphorous; Amox-Zn: amorphous oxide bound zinc; Cryox-Zn: crystalline oxide bound zinc; DAS: days after sowing; DTPA: diethylene Triamine Penta Acetic Acid; MnO₂-Zn: manganese oxide bound zinc; OC-Zn: organically bound zinc; WSEX: water soluble plus exchangeable zinc; MnO₂ Fe: manganese oxide bound iron; OC-Fe: Organically bound iron; WSEX Fe: water soluble plus exchangeable iron.