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Extensin Peroxidase Identification and Characterization in Solanum lycopersicum

Dong, Wen
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1425894387

Abstract Details

2015, Doctor of Philosophy (PhD), Ohio University, Chemistry and Biochemistry (Arts and Sciences).
The regulation of plant cell growth and defense involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Solanum lycopersicum), insolublization occurs by the formation of tyrosylcrosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here, tomato EP candidates were identified using both proteomic and bioinformatic approaches. Bioinformatic screening of the tomato genome yielded eight EP gene candidates, which encoded a putative signal sequence and a protein with a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three, with the lead candidate designated a "CG5". The CG5 open-reading frame from a tomato cDNA was then cloned into a bacterial expression vector to test for EP crosslinking activity. CG5 was expressed in E. coli, fractionated from inclusion bodies, and folded in vitro. Peroxidase activity of CG5 was assayed and quantified by the ABTS (2,2'- zinobis(3-ethylbenzothiazoline-6-sulphonic acid)) assay. Subsequent extensin crosslinking assays showed that CG5 can covalently crosslink authentic tomato P1 extensin and P3-type extensin analogs in vitro supporting the hypothesis that CG5 encodes a tomato EP. Recombinant CG5 was then purified for enzyme kinetics study which gave out a Km of 0.105 mM. A computational 3D model was generated for CG5 with highlighted active site and predicted heme group binding.
Marcia Kieliszewski (Advisor)
Micheal Held (Advisor)
205 p.
Biochemistry; Plant Biology
Hydroxyproline-rich glycoproteins; plant cell wall; extensin; peroxidase; protein crosslinking

Recommended Citations

Citations

  • Dong, W. (2015). Extensin Peroxidase Identification and Characterization in Solanum lycopersicum [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1425894387

    APA Style (7th edition)

  • Dong, Wen. Extensin Peroxidase Identification and Characterization in Solanum lycopersicum. 2015. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1425894387.

    MLA Style (8th edition)

  • Dong, Wen. "Extensin Peroxidase Identification and Characterization in Solanum lycopersicum." Doctoral dissertation, Ohio University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1425894387

    Chicago Manual of Style (17th edition)

ohiou1425894387
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© 2015, all rights reserved.
This open access ETD is published by Ohio University and OhioLINK.