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| Web URL(s): | http://archive.lib.msu.edu/tic/rpr/1994/35247,%20Michigan%20State, Vargas.PDF
Last checked: 11/14/2013
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| Publication Type:
| Report |
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| Material Type: | Manuscript |
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| Monographic Author(s): | Warkentin, D.; Dykema, N. M.; Sticklen, M.; Vargas, J. M. Jr. |
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| Author Affiliation: | Michigan State University |
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| Monograph Title: | Transformation of Creeping Bentgrass With the pHS2 Chitinase Gene: [1994 Annual Research Report], [1994]. |
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| Publishing Information: | [East Lansing, Michigan]: Michigan State University |
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| # of Pages: | 13 |
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| Collation: | [13] pp. |
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| Abstract/Contents: | "More than one thousand creeping bentgrass plants have been putatively transformed for herbicide and insect resistance with the bar and pinII genes. They exhibit resistance to Ignite herbicide (1.2% commercial product containing 200g/L active ingredient of glufosinate ammonium) foliar spray. Molecular analysis of these plants is in progress. The pHS2 chitinase gene cloned from elm chitinase has been successfully manipulated to express in E. coli. When induced with IPTG, E. coli cells with the pGEX-4T-1-pHS2 (plasmid + gene) produced the GST-chitinase fusion protein. This was determined by CDNB assay. The 58kD fusion protein was visible on Coomassie stained SDS Laemli gels. The uninduced cultures containing pGEX-4T-1-pHS2 and both induced and uninduced cultures without pGEX-4T-1-pHS2 did not produce the GST-chitinase fusion protein. Attempts to purify the GST-chitinase fusion protein by adsorption to glutathione-agarose beads were not successful. Therefore, experiments were set up to collect fractions of the bacterial extract at several steps during the purification process. These experiments resulted in discovering that the GST-chitinase fusion protein was not soluble in the buffer used. This, again, was confirmed using SDS Laemli gels. In an effort to remedy this situation, two steps have been taken. They are both currently underway. The first step is to remove the signal peptide currently located at the amino terminal of the protein. This amino acid sequence is 16 amino acids in length and is highly hydrophobic which may be contributing to the insolubility of the GST-chitinase fusion protein. A PCR primer has been designated which will be used to produce a PCR product containing the chitinase gene without the hydrophobic signal peptide. This PCR product will be cloned into a bacterial expression vector. The construct will be used to transform E. coli. Bacterial cultures containing the construct will be induced with IPTG and tested to determine if they contain soluble chitinase with antifungal activity. The second step underway is transformation of tobacco with the pHS2 chitinase gene. Tobacco is relatively quick and easy to transform via Agrobacterium. This successful transformation will give us information about the plant's ability to produce active chitinase from pHS2. If tobacco can successfully produce pHS2 chitinase with antifungal activity, this would indicate that bentgrass plants will likely do the same. At that point it is expected that no further manipulation of the pHS2 chitinase genes will be necessary before the transformation of bentgrass plants. Upon successful transformation of tobacco, leaf extracts will be used in bioassays to test for antifungal activity. Antifungal bioassays have been attempted. However, due to the insolubility of the GST-chitinase fusion protein produced in bacteria, no activity has yet been observed." |
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| Language: | English |
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| References: | 5 |
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| See Also: | See also related summary article "Control of bentgrass pathogenic fungi dollar spot, brown patch and pythium blight using chitinase" 1994 Turfgrass Research Summary [USGA], 1994, p. 40-41, R=35247. R=35247 |
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| Note: | Also appears as pp. 00425-00437 in the USGA Turfgrass Research Committee Reporting Binders for 1994.
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| ASA/CSSA/SSSA Citation (Crop Science-Like - may be incomplete):
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http://archive.lib.msu.edu/tic/rpr/1994/35247,%20Michigan%20State, Vargas.PDF
Last checked: 11/14/2013
Requires: PDF Reader |
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