Cloning and Characterization of Sdga Gene Encoding .ALPHA.-Subunit of Heterotrimeric Guanosine 5'-Triphosphate-Binding Protein Complex in Scoparia dulcis

Abstract

A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding a a-subunit of heterotrimeric guanosine 5Ј-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein a a-subunits from various biological sources suggested that, unlike in animal cells, classification of Ga a-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Ga a-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.Key words a-subunit; guanosine 5Ј-triphosphate-binding protein; heterotrimer complex; Scoparia dulcis Biol. Pharm. Bull. 31(11) 2150-2153 (2008) © 2008 Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail: kurosaki@pha.u-toyama.ac.jp (G-Biosciences) according to the instruction manuals, and the restriction digests were prepared using XbaI, XhoI, EcoRI, or EcoRV (Takara). They were electrophoresed on a 0.8% agarose gel, and the separated DNA fragments were transferred onto an Immobilon-NY ϩ (Millipore). The translatable region of Sdga including stop codon (1152 nucleotides) was amplified by PCR and was directly labeled with AlkPhos Direct Labeling and Detection System (GE Healthcare Bio-Science) to be used as the probe. The membrane was hybridized with the probe at 55°C for 24 h in a solution containing 6ϫSSC, and, after appropriate washings, the filters were dried and exposed to an X-ray film at room temperature for 3 h. Expression of Sdga GeneThe expression level of Sdga was analyzed semi-quantitatively using RT-PCR. The leaves of S. dulcis were detached from the middle part of the stem, and incubated with MJ (100 mmol l Ϫ1 water, Wako Pure Chemicals) in Petri dishes at 26°C under constant illumination according to the method described previously. 20) Control treatments received only water instead of MJ. At regular intervals, the treated leaves were harvested and total RNA was isolated according to the method described above. Aliquots of RNA solutions (approximately 0.5 mg RNA equivalent) were subjected to RT reaction using Transcriptor First Strand cDNA Synthesis Kit (Roche), and then, PCR was carried out with the primer pair, 5Ј-ATG CGG TGG ATG CTA ATC November 2008 2151 Fig. 1. Alignment of Amino Acids Sequences of a-Subunits of GTP-Binding Protein Complex from Various Biological SourcesA cDNA clone encoding the a-subunit of GTP-binding protein, Sdga, was isolated from S. dulcis, and the deduced amino acid sequence was compared with the proteins from various biological sources (Arab, Arabidopsis thaliana; Rice, Oryza sativa...