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Isolation and identification of -cyanoalanine synthase from etiolated rice (Oryza sativa) seedings

by Cheung, Yee-wai

Abstract (Summary)
(Uncorrected OCR) Abstract of thesis entitled ISOLATION AND IDENTIFICATION OF 0-CYANOALANINE SYNTHASE FROM ETIOLATED RICE {Oryza sativa) SEEDLINGS Submitted by Cheung Yee Wai for the degree of Master of Philosophy at The University of Hong Kong in August 2004 /3-Cyanoalanine synthase (CAS) is a key enzyme in the ethylene biosynthetic pathway for the detoxification of the co-product cyanide. Though CAS has been purified and identified in other plant species, the identification and molecular cloning of CAS in rice {Oryza sativa) has not yet been described. In this study, CAS in 7-day old etiolated rice seedlings was purified 250-fold by chromatography and preparative electrophoresis. The partially purified CAS had a native molecular mass of 60,000 daltons and possessed only CAS activity, unlike the previously reported enzymes which also possess cysteine synthase (CS) activity. This suggests that it is a newly discovered protein in the CAS sub-family. The purified CAS was further identified by 2-dimensional electrophoresis (2-DE) and quadrupole time-of-flight mass spectrometry (Q-TOF-MS) analysis. A unique internal tryptic peptide (LIVTVLPSLGER) was found to be associated with the rice CAS activity, and a corresponding 1134 bp cDNA open reading frame (OSCAS) was identified in the rice gene bank database. The predicted enzyme, OSCAS, consists of 378 amino acid residues was predicted. Molecular cloning analysis of the OSCAS cDNA was conducted by reverse-transcription polymerase chain reaction (RT-PCR) of mRNA, which extracted from rice seedlings; and OSCAS protein expression was achieved by using the GST fusion protein system. The expressed fusion protein was found mainly in the insoluble inclusion bodies of E. coli. The expression patterns of OSCAS in 7-day old etiolated rice seedlings and 10-day old green rice seedlings were investigated by Northern blot analysis and native PAGE. Results showed that the OSCAS transcript and OSCAS activity in etiolated seedlings were significantly induced by a synthetic auxin: 2, 4-dichlorophenoxyacetic acid (2,4-D). Surprisingly, expressions of the OSCAS and OSCAS activity in both etiolated and green seedlings were suppressed by exogenous ethylene, indicating that CAS in rice is subtly regulated by ethylene biosynthesis and signal transduction pathways.
Bibliographical Information:

Advisor:

School:The University of Hong Kong

School Location:China - Hong Kong SAR

Source Type:Master's Thesis

Keywords:plant enzymes ethylene synthesis molecular cloning rice genetics

ISBN:

Date of Publication:01/01/2004

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