Journal of Applied Biosciences (J. Appl. Biosci.) [ISSN 1997 - 5902]
Volume 10(2): 538 - 546. Published October 7, 2008.
Design, construction and cloning of pCAMBIA-MiAMP1 vector for enhancing disease resistance in plants using Agrobacterium-mediated transformation
Ghasemi Bezdi Kamal*^, Sheveloukha Victor Stepanovich^, Karlov Gennady Illich^
*1Cotton Research Institute of Iran, Beheshti St, P.O.Box 49175-483, Gorgan, Iran; 2Russian State Agrarian University – MTAA (named after K. A. Timiriazev), Department of Agricultural Biotechnology, Timiriazevskaya St., No 49, Moscow 127550, Russia.
^Corresponding author e–mail:kghasemibezdi@yahoo.com
ABSTRACT
Objective: To design, construct and clone a pCAMBIA-MiAMP1 vector that can be used to enhance disease resistance in plants through Agrobacterium-mediated transformation.
Methodology and results: The cDNA sequence encoding MiAMP1 antimicrobial peptide as a defense resistance gene, which had been cloned into the cloning site of pGEM-T T-vector, was obtained from Australia. The pGEM-T-MiAMP1 was transformed into E.coli and the identification of transformed clones among antibiotic resistant bacteria carried out by lacZ expression using blue/white screening system. The presence of MiAMP1 gene was confirmed using PCR analysis with primers M13, restriction enzymes NcoI and BamHI and DNA sequencing. Based on the sequence, the orientation and restriction map of target gene in plasmid was defined. After designing the specific PCR primers flanking the coding region of the MiAMP1 gene and amplifying the complete cDNA with additional restriction sites, either PCR product, or the binary vector pCAMBIA1305.1 were digested with restriction enzymes NcoI and BglII. Subsequently, the insert and the vector were ligated with T4 DNA ligase to produce binary vector pCAMBIA-MiAMP1 for genetic transformation of plants. This construction contains the full coding region of the MiAMP1 antimicrobial peptide and is flanked at its 5' end by the strong constitutive promoter of CaMV35S and at its 3' end by the polyadenylation sequence of NOS polyA. The designed expression vector also contains other elements that are useful for plant transformation such as a kanamycin resistance gene (npt II) and a hygromycin resistance gene (hpt II) that can be used as markers for selecting transformed plants, while the GUSPlusä reporter gene contains the intron. The presence of MiAMP1 gene in the pCAMBIA-MiAMP1 construction was confirmed among antibiotic resistant colonies by PCR method, restriction enzymes analysis and DNA sequencing. The designed construction was transferred into Agrobacterium tumefaciens strain AGL0 by tri-parental mating method with the helper plasmid pRK2013.
Conclusion and application of findings: pCAMBIA-MiAMP1 construction can be used to genetically transform plants to enhance resistance to fungi e.g. Leptosphaeria maculans and Sclerotinia sclerotiorum. Currently, it is being used in Agrobacterium-mediated transformation of canola and sunflower.
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