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United States Department of Agriculture

Agricultural Research Service

Title: Cloning and Construction of Single-Chain Variable Fragments (Scfv)to Cucumber Mosaic Virus and Production of Transgenic Plants

Authors
item Aebig, Joan - FORMER ARS EMPLOYEE
item Albert, Henrik
item Zhu, B - FORMER ARS EMPLOYEE
item Hu, J - UNIV OF HAWAII
item Hsu, Hei Ti

Submitted to: Acta Horticulture Proceedings
Publication Type: Proceedings
Publication Acceptance Date: May 16, 2005
Publication Date: October 31, 2006
Citation: Aebig, J.A., Albert, H.H., Zhu, B.L., Hu, J.S., Hsu, H.T. 2006. Cloning and construction of single-chain variable fragments (SCFV) to cucumber mosaic virus and production of transgenic plants. Acta Horticulture. 722:129-136.

Interpretive Summary: Cucumber mosaic virus (CMV) causes economically important diseases in many crops including cereals, vegetables, and ornamentals. Crop losses due to the disease are tremendous. The virus is naturally transmitted by many species of aphids and by vegetative propagation. High quality and productivity of floriculture crops can only be guaranteed by extensive control of pests and availability of virus-free propagation materials. We have previously produced serogroup-specific and virus-specific mouse monoclonal antibodies to CMV for taxonomic and diagnostic purposes. Furthermore, these antibodies were found to neutralize virus infectivity and block virus transmission by aphids making them candidates for engineering CMV resistance in plants. In this paper, immunoglobulin heavy and light chain variable regions were cloned from hybridoma cells secreting CMV antibodies. Using a peptide linker, both heavy and light chain variable regions were constructed into single-chain variable fragments (ScFvs). The constructs were transferred into Nicotiana benthamiana leaf tissue and transgenic plants containing CMV-ScFv were produced. Resistance to CMV infection was evaluated by mechanically inoculating the plants with the virus. The content is intended for use ny readers with knowledge of basic plant pathology, virology and molecular biology. It is revelant to the National Plant Health Program 303.

Technical Abstract: The variable regions of heavy and light chain genes were cloned from the RNA of mouse hybridoma cells that produce monoclonal antibodies specific to Cucumber mosaic virus (CMV). The cloned genes were constructed into single-chain variable fragments (scFv) in combination with various promoters and transferred into Nicotiana benthamiana leaf tissue using Agrobacterium tumefaciens. Transgenic plants were generated and selected using the bar gene that confers resistance to phosphinothricin. Presence of the transgene was verified by sequencing scFv-specific PCR amplicons from genomic plant DNA. Resistance to CMV infection was evaluated by mechanically inoculating plants with purified CMV. Several R0 transgenic plants exhibited some degree of disease resistance: showing no symptoms reduced symptoms or delayed symptom development. In subsequent challenge inoculations of R1 plants with lower dilutions of inoculum, some plants showed resistance when compared with non-transformed plants, but, overall, results were variable. For each of the four promoters tested, three or four transgenic plants were generated exhibiting some degree of disease resistance.

Last Modified: 11/28/2014
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