Hypertension is a common chronic disease and causes serious damages to human health. Medicine therapies are effective and widely used in controlling and curing hypertension worldwide. However, these medical treatments hold many associated problems, such as high costs and side effects. Rice is the principal food for most population in Asia and widely consumed in other continents around world. Saikusa et al. (1994) reported that water soaking brought about the remarkably changes of the constitute and content of free amino acid in the rice 'Koshihikari' kernel, among which the increase of gamma-aminobutyric acid (GABA) content was the most significant. It was reported that GABA had a function to reduce blood pressure (Takahashi et al., 1955), therefore the rice grain rich in GABA could be expected as the attractive candidate of function food for hypertension patient. Glutamate decarboxylase (GAD, EC 4.1.1.15), which catalyzed the irreversible decarboxylation of glutamate, was believed to be the key enzyme in GABA synthesis (Huang et al., 1990). Two GAD cDNA (OsGAD1-2) had been isolated in rice genome (Akama et al, 2001).

In this study, we have cloned a novel glutamate decarboxylase cDNA of rice (Oryza sativa L), OsGAD3 (GenBank accession No. AY187941 ) by the combination of bioinformatics and RT-PCR approaches. Sequence analysis reveals that OsGAD3 contains a 1476 bp ORF that encodes a 492 amino acid peptide with a molecular mass of 56.0 KDa. Comparison of the deduced amino acid sequences shows that OsGAD3 is closely related to the glutamate decarboxylase from dicotyledon plant other than OsGAD1 and OsGAD2 (Fig. 1).

The entire cDNA ORF coding for the OsGAD3 protein was inserted into a pET30a(+) expression vector, and the expression of the recombinant His-tag fusing protein was induced by the addition of 1mM IPTG. The recombinant proteins were detected in the insoluble fraction extracted from IPTG-treated E. coli carrying the the recombinant plasmid pET::OsGAD3 (Fig. 2), but not in the soluble fraction from the same bacteria (data not shown), suggesting that the recombinant protein mainly exist in inclusion body. The molecular mass of recombinant protein is about 61 KD, which is consistent with the total molecular mass of deduced OsGAD3 protein and histidine tag. Furthermore, using a spectrophotometric method as described by Johnson et al (1997), the GAD activity of catalyzing the conversion of glutamic acid to GABA was separately tested in total (infractionated) extracts of cultures carrying control plasmid, and insoluble (pellet) fractions of cultures carrying the recombinant plasmid before and after IPTG induction. As shown in Fig. 3, the activities in insoluble fractions of induced cultures carrying the recombinant plasmid were about 5-times higher than those in the other E. coli cultures. This result confirmed that OsGAD3 cDNA encoded a functional GAD enzyme.

Moreover, semi-quantitative RT-PCR was carried out to detect the gene expression of three glutamate decarboxylase in steeped rice grain (Fig. 4). The results demonstrated that OsGAD3 and OsGAD1 were the major transcripts with overlapping but slightly distinct expressing

pattern. Between them, OsGAD3 showed higher expression level. Therefore it is speculated that OsGAD3 may play a dominant role associated with GABA accumulation and the physiology functions in the early stage of seed imbibition. In marked contrast, OsGAD2 was almost undetectable until coleoptile appeared when its expression started to rise. It implied that perhaps OsGAD2 performed a unique function during germination and seedling development. We also found that the increase of whole OsGADs transcripts were parallel with the accumulation of GABA in rice grain during water soaking, suggesting the level of GABA was in partly regulated at transcriptional process.

The plant expression vector for OsGAD3 was constructed under the control of 35S promoter and transformed into Arabidopsis plant via the method of dipping flower. The transgenic plants expressing a rice GAD exhibited higher GAD activities and GABA levels than wild plants. The results provided the evidence to the possibility of increase GABA content by metabolic engineering.

References

Saikusa, T., T. Horino and Y. Mori, 1994. Distribution of free amino acids in the rice kernel and kernel fractions and the effect of water soaking on the distribution. J. Agri. Food Chem. 42:1122-1125.

Takahashi, H, M. Tiba, M. Iino and T. Takayasu, 1955. The effect of gamma-aminobutyric acid on blood pressure. Jpn. J. Physiol. 5: 334-341.

Huang, W.M., R.F. Lori, E. Wu and J.Y. Wu, 1990. Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase. Proc. Natl. Acad. Sci. 87: 8491-8495.

Akama, K., T. Akihiro, M. Kitagawa and F. Takaiwa, 2001. Rice (Oryza sativa) contains a novel isoform of glutamate decarboxylase that lacks an authentic calmodulin-binding domain at the C-terminus. Biochim. Biophys. Acta 1522: 143-150.

Johnson, B.S., N.K. Singh, J.H. Cherry and R.D. Locy, 1997. Purification and characterization of glutamate decarboxylase from cowpea. Phytochemistry. 46: 39-44.