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Title: ISOLATION, CLONING AND OVEREXPRESSION OF THE COTTON RUBISCO ACTIVASE GENE.

Author
item Salvucci, Michael
item VAN DE LOO, FRANK - UNIV. OF KENTUCKY

Submitted to: National Cotton Council Beltwide Cotton Conference
Publication Type: Proceedings
Publication Acceptance Date: 6/1/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: In the process of photosynthesis, plants convert light into the chemical energy that is used to synthesize sugars and other foodstuffs. The first and rate-limiting step in photosynthesis involves the fixation of atmospheric carbon dioxide by the enzyme Rubisco. The activity of Rubisco is regulated by another enzyme called Rubisco activase. Rubisco activase controls the overall process of photosynthesis by altering the activity of Rubisco in response to environmental conditions. Mounting evidence indicates that Rubisco activase is a key control point limiting photosynthesis under conditions of environmental stress. This paper reports the isolation of the gene for Rubisco activase from cotton. Information about the gene for cotton Rubisco activase provides detailed information on the structure of this important enzyme. Knowledge of the structure of Rubisco activase eventually can be used to make changes that improve the activity of the enzyme. An improved Rubisco activase could be used to increase the photosynthetic efficiency of cotton, particularly under adverse environmental conditions.

Technical Abstract: Activation of Rubisco, the primary carboxylating enzyme in photosynthesis, is promoted in vivo by the enzyme Rubisco activase. As a first step in investigating Rubisco activation in cotton, we screened a cotton (Gossypium hirsutum L.) cDNA library to isolate the cotton activase (rca) gene. Restriction and sequence analysis of positive clones indicated that there were three classes of cotton rca cDNAs. One of these classes encodes for a mature protein of 380 amino acids (42357 Da), while the other two encode for mature proteins of 419 amino acids (46590 Da). These results were consistent with Southern analysis which indicated that there are multiple cotton activase genes and with Western blot analysis which showed the presence of two activase polypeptides in cotton leaf tissue. The deduced amino acid sequences of the cotton rca clones shared 80 to 89% identity with activases from other higher plants. One of the cotton activase clones was overexpressed in E. coli, enabling the production of large amounts of soluble cotton activase protein for functional analysis.