Vermont Agricultural Experiment Station Researchers' Participation in Multistate Research
(July 2003)
MULTISTATE PROJ NO: NCR-170
ACCESSION NO: 0189948
PROJ NO: VT-PS-00807
START: 01 OCT 2001 TERM: 30 SEP 2004 FY: 2002
INVESTIGATOR: Aleong, J.
OBJECTIVES: Travel grant to attend NCR-170
APPROACH: Travel grant to attend NCR-170
MULTISTATE PROJ NO: NC-1009
ACCESSION NO: 0194322 SUBFILE: CRIS
PROJ NO: VT-AS-00920
START: 01 OCT 2002 TERM: 30 SEP 2007
INVESTIGATOR: Knapp, J.
METABOLIC RELATIONSHIPS IN SUPPLY OF NUTRIENTS FOR LACTATING COWS (NC-185)
OBJECTIVES: 1) To quantify properties of feeds that determine the availability of nutrients critical to milk production. 2) To quantify metabolic interactions among nutrients that alter synthesis of milk. 3) To use these quantitative relationships to challenge and refine computer-based nutrition systems for dairy cattle.
APPROACH: The long term goal of this laboratory is to increase our understanding of nutrient digestion, absorption and utilization in ruminants. Glucose and lipid metabolism are critical to both growing and lactating ruminants, and the development of research techniques to acquire more accurate, detailed and quantitative knowledge in this area is crucial to advancing ruminant nutrition and applied animal feeding. An cDNA microarray approach will be utilized to quantify the relationships between milk yield and gene expression. The microarrays will be validated with independent methods.
NON-TECHNICAL SUMMARY: While milk production per cow is 2.5 times what is was 30 years ago, there is no reason to believe that milk production in dairy cattle has been maximized. Also, while production levels have improved due to genetic selection, our understanding of the underlying biological mechanisms of how this has been achieved is limited. Furthering such understanding will allow additional improvements in genetic selection and better nutritional and health management strategies to sustain the viability of dairy farming in Vermont, the NorthEast, and the rest of the U.S.
MULTISTATE PROJ NO: NC-209
ACCESSION NO: 0193462
PROJ NO: VT-AS-00906
START: 01 OCT 2002 TERM: 30 SEP 2005
INVESTIGATOR: Kerr, D. E.
INTERPRETING CATTLE GENOMIC DATA: BIOLOGY, APPLICATIONS, AND OUTREACH
OBJECTIVES: 1) To identify and characterize genes and gene products that influence reproduction, disease resistance, milk and meat production efficiency, composition and quality. 2) To develop methods for statistical mapping and characterization of economic trait loci using granddaughter, daughter, and other designs. 3) To develop methods to use molecular markers for quantitative traits in applied breeding programs.
APPROACH: One of the major goals of the proposed experiments is to use microarray technology to evaluate infection responsive genes in the bovine mammary gland. Unfortunately the variability in distribution of infection within a mammary gland negates using tissue biopsies as a source of representative RNA. Thus, an in vitro model will be developed in which cultured acini, isolated from lactating tissue, will be infected during a short-term culture period. A problem here is that even with short-term culture the acini will likely begin to develop an involution phenotype. Therefore in development of the model the degree of involution will first be determined. This will be followed by subsequent experiment where infection will be superimposed. Gene expression profiling will be performed with a microarray technique that simultaneously compares the expression profiles of two different RNA samples. The proposed experiments seek to determine genes abundantly expressed (or repressed) in samples from involuting or infected cells as compared to normally lactating cells. Analysis of down-regulated genes is outside the scope of the current proposal. The identified genes will then be evaluated for their potential to serve as regulatory elements to drive novel antibacterial proteins as a means to enhance resistance to mastitis. Initial evaluation will consist of northern blot confirmation of the expression profile. Others have found that the array data itself is not overly sensitive, and thus the northern confirmation is required. The next step will be to clone, or obtain from colleagues, the gene regulatory region. The arrays are assembled from mRNA and thus it is often the case that the regulatory region has not been cloned. In this case the appropriate cDNA will be used to screen a genomic library for the gene of interest. Ultimately the regulatory region of the identified gene will be linked to a structural region encoding an antibacterial protein such as lysostaphin. Functionality of the gene construct will be confirmed by transfection into cultured cells, then the gene construct will be inserted into transgenic mice for in vivo evaluation. The generation of transgenic mice is likely beyond the scope of the current proposal, but we have a proven track record in the ability to complete such a project.
NON-TECHNICAL SUMMARY: Infection of the bovine mammary gland is the most costly disease of dairy cattle. Antibiotic therapy is currently a major part of management strategies to prevent or cure mastitis. Our laboratory is seeking to generate transgenic animals that have enhanced resistance to mastitis, an thus would require less antibiotic treatment. The mammary glands of these animals produce new antibacterial enzymes that target mastitis causing bacteria.
MULTISTATE PROJ NO: NE-183
ACCESSION NO: 0182918
PROJ NO: VT-PS-00614
START: 01 OCT 1999 TERM: 30 SEP 2004
INVESTIGATOR: Garcia, M. E.; Berkett, L. P.
OBJECTIVES: To remain competitive, Vermont apple growers have to analyze the profitability of the various components of their current orchard operation and plan for the future. Cultivar and rootstock selection is a crucial decision for apple growers that will impact the farm's competitiveness and profitability for many years.
APPROACH: Multidisciplinary evaluation of new apple cultivars and rootstocks including an examination of the cold hardiness of certain cultivars and an assessment of disease susceptibility using an intergrated pest management approach will provide valuable information that Vermont apple growers need to make this critical decision. This research is an integral part of NE-183 and NC-140 regional research projects. Established protocols will be forwarded for data collection and analysis.
MULTISTATE PROJ NO: NC-140
ACCESSION NO: 0192182
PROJ NO: VT-PS-00810
START: 01 OCT 2001 TERM: 30 SEP 2007
INVESTIGATOR: Garcia, M. E.; Berkett, L. P.
OBJECTIVES: 1. To evaluate the performance of pome-and-stone-fruit rootstocks in various environments and under different management systems. 2. To assess and improve asexual propagation techniques of pome-and stone-fruit rootstocks. 3. To improve the ability to identify and identify pome-and stone-fruit rootstocks through morphological, biochemical, and genetic differences. 4. To develop new and better pome- and stone-fruit rootstocks through breeding and genetic engineering, and to acquire new rootstocks from breeding programs in other parts of the world. 5. To determine biotic and abiotic stress tolerances of pome- and stone-fruit trees in relation to new and existing rootstocks.
APPROACH: Tree maintenance and data collection for the 1999 planting of the NC-140 Cornell/Geneva Apple Rootstock Evaluation continues at the University of Vermont Horticultural Research Center in So. Burlington, VT. The trees were trained, pruned and maintained according to protocol developed by the Principal Leaders. Data collected include number of flower clusters, number of Rootsuckers, number of Burrknots, number of fruit on tree, weight of Fruit On Tree, number of Drops, weight of Drops, and TCSA.
MULTISTATE PROJ NO: NC-1004
ACCESSION NO: 0193461
PROJ NO: VT-AS-00905
START: 01 OCT 2002 TERM: 30 SEP 2005
INVESTIGATOR: Hovey, R. C.
GENETIC AND FUNCTIONAL GENOMIC APPROACHES TO IMPROVE PRODUCTION AND QUALITY OF PORK
OBJECTIVES: The following objectives of this multistate project are: 1) Further understand the dynamic genetic mechanisms that influence production efficiency and quality of pork. 2) Discover genetic mechanisms controlling animal health in pork production. The current proposed research will contribute to Objective 1 of this project. To this end the following specific objectives are proposed: Objective 1. The first objective of this project will be to clone the various isoforms of the pPRLR. Objective 2. In objective 2 we will examine the level and distribution of PRLR mRNA(s) in various tissues of pigs. Objective 3. In objective 3 we will determine the functions and characteristics of pPRLR isoforms.
APPROACH: Objective 1. In this objective we will clone and define the molecular structure of various pPRLR isoforms. Using current information we will first amplify the intermediary cDNA sequence from reverse-transcribed RNA (from pig mammary gland) by PCR to provide near full-length cDNA for the long pPRLR. This will then be cloned and sequenced (at the Vermont Cancer Center) to provide sequence for the transmembrane and adjacent intracellular regions of this isoform. In objective 2 we will determine the distribution and expression of pPRLR in developing female pigs that will utilize results from objective 1, and will complements findings from objective 3. Information obtained from this objective using molecular and biochemical techniques will provide data to indicate potential physiological functions of pPRLR, specifically during growth and female reproductive development in pigs.Objective 3. In objective 3 we will determine the functional characteristics of various pPRLR isoforms we have identified in objective 1. Whereas data from objective 2 will provide important information concerning the expression and distribution of pPRLR, results from this objective using experiments performed in vitro will define their biological functions. Experiments for this objective will be conducted in year 3 of the project. From objective 1 we will have access to full-length coding sequence for various forms of the pPRLR.
NON-TECHNICAL SUMMARY: The function and mechanism of prolactin action in the pig is unknown. This project will identify the mechanism by which prolactin signals to target cells in pigs.
MULTISTATE PROJ NO: NC-1010
ACCESSION NO: 0197004
PROJ NO: VT-AS-00922
START: 01 OCT 2002 TERM: 30 SEP 2005
INVESTIGATOR: Zhao, F.
APPROACH: Animals, Tissue Sample Collection and RNA Isolation: Tissue samples have been collected at local USDA slaughter houses from lactating dairy cows, shipped from the University of Vermont Farm. The tissue samples were collected immediately after the animals were euthanized. Following excision of the tissue, it was either processed for immunocytochemical analysis as described below or rapidly frozen in liquid nitrogen and stored at -80C. The total RNAs or poly(A) RNAs will be obtained from several sources. High quality poly(A) RNAs from bovine brain, heart, kidney, liver and spleen will be purchased from the BD Biosciences Clontech and used for gene cloning. The RNA used for gene expression assays will be isolated using TRIzol reagents (Invitrogen) from the frozen bovine tissues taken at local slaughter houses. Gene Cloning: GLUTs 8-10 and SGLTs 2-3 will be cloned by 5' and 3' RACE (Rapid Amplification of cDNA Ends) using Clontech SMARTTM RACE cDNA Amplification kit with the GeneAmp PCR System 9700 that was purchased using the applicant's startup funds. The individual transporters will be cloned from different bovine tissues in which the GLUT/SGLT has been shown the strongest expression in other species. The primers used in RACE will be generated from the corresponding bovine EST sequences in GenBank. Alternately, degenerate oligo primers will be generated in conserved regions of the known transporter cDNA sequences of other species. After RACE products have been sequenced, the full-length cDNA of each individual transporter will be generated by RT-PCR using primers designed from the extreme 5' and 3' ends of the cDNA. The cDNA clones for each transporter will be sequenced using ABI Automatic DNA Sequencer. Expression of GLUTs & SGLTs in Bovine Tissues: Expression of mRNA of GLUTs 8-10 and SGLTs 2-3 in bovine tissues will be analyzed using Northern blots and each transporter's cDNA clone as a probe. An alternative strategy will be use more sensitive RT-PCR or Taqman methods. The procedures of Northern blot and RT-PCR will follow the applicant's previously developed methodologies (Zhao et al., 1996a). The Taqman assay will follow the manufacturer's instructions (ABI 7700). Localization of SGLT1 in Bovine Mammary Gland: The cellular localization of SGLT1 will be analyzed by light microscopy immunolabelling as described by Zhao et al. (1996a). Tissue samples from bovine mammary gland are fixed in 4% (w/v) paraformaldehyde in PBS for 4 h at 4C, washed in PBS, and immersed in 0.5 M sucrose in PBS overnight. Tissue blocks are mounted on specimen holders and frozen in liquid nitrogen. Sections (10 um) are cut and thaw-mounted on the surface of gelatin-coated slides. Immunocytochemical staining is performed according to the peroxidase-antiperoxidase procedure, with 3',3-diaminobenzidine (DAB) as cosubstrate. The electron microscopy immunolabeling will be performed using the procedure described by Camps et al. (1994). Test Commercial Antibodies for the use in bovine tissues : Western Blotting will be performed as described before (Zhao et al., 1996a).
NON-TECHNICAL SUMMARY: Glucose uptake by the milk-producing cells in the mammary gland is the rate-limiting step for milk production because glucose is the major precursor of milk lactose which controls milk yield. This project is to clone the glucose transporters in bovine tissues which provide the key role in maintaining glucose homeostasis in lactation.
MULTISTATE PROJ NO: NE-1012
ACCESSION NO: 0195163
PROJ NO: VT-AE-00803B
START: 01 OCT 2002 TERM: 30 SEP 2007
INVESTIGATOR: Kolodinsky, J. M.
APPROACH: This project will use both survey research with consumers and individual indepth interviews with food buyers in grocery stores. Both statistical and qualitative methodologies will be used to analyze the data.
NON-TECHNICAL SUMMARY: The issue of consumer acceptance of biotechnology is reaching a critical juncture. There is a gap in information sharing between industry, sellers, and the final consumer. This study will document emerging trends in attitudes, knowledge and behavior related to genetical modification of food and will estimate willingness to pay for information about agricultural biotechnology.
MULTISTATE PROJ NO: S-301
ACCESSION NO: 0195164
PROJ NO: VT-PS-00921
START: 01 OCT 2000 TERM: 30 SEP 2005
INVESTIGATOR: Brownbridge, M.; Soto-Adames, F.; Harper, W. S.
APPROACH: Impact of Beneficial Microbes and Insect-Resistant Transgenic Corn on Non-Target Soil Microarthropods. Integration of biologically-based pest management techniques into crop protection systems is critical to the long-term well-being of US agriculture. However, we cannot assume that biological controls are themselves without risk, and we must proactively evaluate effects of microbial control agents and insecticidal toxins expressed by transgenic plants on key indicator species to ensure minilal environmental impact. Special attention must be paid to effects on important soil fauna because of the possible accumulation and/or multiplication of microbes and toxins in this environment. Collembola play a vital role in the removal, breakdown and re-cycling of crop residues, and are abundant in healthy soils; they represent an ideal non-target indicator species. This research initiative will allow us to begin to identify and quantify potential ecological side-effects of these biological control practices on a scientific basis and will help define future research needs in this area. In laboratory and field tests, entomopathogenic and antagonistic fungi and bacteria used in crop protection, e.g., Beauveria bassiana, Metarhizium anisopliae, Bacillus thuringiensis, Trichoderma harzianum, will be presented to two species of Collembola using a novel feeding assay; effects on survival, longevity and fecundity will be determined. Effects of Bt corn (root toxins and crop residues) on diversity and abundance of Collembola and other microarthropods will be determined by season-long sampling of soils planted with transgenic and non-transgenic corn. Microarthropods will be extracted by Berlese funnels, and species diversity and abundance compared over three cropping cycles, from pre-planting through post-harvest. Finally, the stability of the Bt toxin during composting (two composting systems will be tested) and silage fermentation will be determined and effects of the composted material on Collembola evaluated. Toxin stability during these processes is not well documented; break-down of the toxin will avoid any potential `downstream' side-effects on non-target organisms.
NON-TECHNICAL SUMMARY: There are clear benefits to using biopesticides and genetically-modified plants in a pest management program but proactive steps must be taken to ensure they have minimal impact on biodiversity. The current project aims to quantify effects of selected microbial pesticides and transgenic corn on Collembola; these soil microarthropods are essential components of a healthy soil biota but side-effects of crop protectants on these organisms have been poorly documented.
North Central