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Positive-selection and ligation-independent cloning vectors for large scale in Planta expression for plant functional genomics

Cited 14 time in Web of Science Cited 15 time in Scopus
Authors
Oh, Sang-Keun; Kim, Saet-Byul; Yeom, Seon-In; Lee, Hyun-Ah; Choi, Doil
Issue Date
2010-12
Publisher
Korean Society for Molecular and Cellular Biology
Citation
Molecules and Cells Vol.30 No.6, pp. 557-562
Keywords
복합학ccdB genelarge-scale plant expression vectorsligation-independent cloning
Abstract
Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligationindependent cloning (LIC) vectors derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC, and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes, alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3' to 5' exonuclease activity in the presence of only one dNTP #dGTP for the inserts and dCTP for the vector#. To make recombinants, the vector plasmid and the insert PCR fragment were annealed at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100% efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance #R# genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic studies in plants.
ISSN
1016-8478 (print)
0219-1032 (online)
Language
English
URI
https://hdl.handle.net/10371/83453
DOI
https://doi.org/10.1007/s10059-010-0156-2
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Plant Science (식물생산과학부)Journal Papers (저널논문_식물생산과학부)
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