MOLECULAR CLONING AND CHARACTERIZATION OF GLUTAMATE DECARBOXYLASE CDNA FROM THE GIANT-EMBRYO ORYZA SATIVA

A full-length cDNA encoding glutamate decarboxylase (designated as OsGAD3), which catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA), was isolated from the GABA-rich giant-embryo Oryza sativa (Shangshi Jing 315). The full-length cDNA of OsGAD3 (SSJ315) has a 1479 bp open reading frame (ORF) encoding a protein of 492 amino acid residues. The deduced protein had an isoelectric point (pI) of 5.72 and a calculated molecular weight of 56.1 kD. Sequence comparison showed that OsGAD3 (SSJ315) matches the glutamate decarboxylases of other plant species reported previously. Analysis of the structural features of the C-terminal portions of plant GADs revealed that OsGAD3 (SSJ315) has the typical CaM-binding domain (CaMBD) in the C-terminal region as most other plant GADs. Evolution analysis showed that plant GADs are conserved in the process of evolution. The cloning and characterization of the OsGAD3 (SSJ315) gene will enable us to use OsGAD3 to enhance GABA production in O. sativa (SSJ315) by metabolic engineering in the near future.


INTRODUCTION
Hypertension is a common chronic disease and is regarded as one of the most critical risk factors causing serious damage to human health (Liu et al., 2005;Akama et al., 2009).Reports indicate that nearly one billion people are now suffering from hypertension in the world (Kearney et al., 2005).Up to now, patients with severe hypertension mainly depend on different kinds of blood pressurelowering drugs (Liu et al., 2005;Akama et al., 2009), but they have several shortcomings, including the high cost of drugs for long-term treatment, development of drug dependence in patients and side effects (Liu et al., 2005;Akama et al., 2009).Therefore, it is necessary to develop a new effective way for the treatment of hypertension.
Recently, foods or diets that contain bioactive substances with anti-hypertensive effects have received more attention (Akama et al., 2009).Gamma-aminobutyric acid (GABA), as non-protein amino acid that is a well-characterized inhibitory neurotransmitter for animals with hypotensive and analgesic properties (Erlander et al., 1991;Hayakawa et al., 2004).Therefore, food-based interventions to increase the availability of GABA-rich foods and their consumption have been suggested as an alternative way for the treatment of hypertension (Tang et al., 2009).For example, several giant-embryo rice varieties or lines which are rich in GABA (which has the function of reducing blood pressure) such as Haiminori, Giant-embryo No.1, 6601, W025, Gama-1, TgeA and 98-14geA, have been selected by mutant or cross introgression recently (Zhang et al., 2007).
More recently, the development of plant metabolic engineering provides a possibility for the further improvement of GABA content by transferring the key genes involved in the GABA biosynthetic pathway into GABA-rich giant-embryo rice.However, this significantly relies on the detailed understanding of the pathway for GABA biosynthesis, the enzymes catalyzing the reaction chain, especially the ratelimiting steps and the genes encoding these proteins.
GABA is metabolized mainly via a short, threeenzyme pathway known as the GABA-shunt because it bypasses two successive steps of the tricarboxylic acid (TCA) cycle.Glutamate decarboxylase (GAD,EC 4.1.1.15)catalyzes the conversion of glutamate to GABA; then GABA transaminase (GABA-T) catalyzes the conversion of GABA to succinic semialdehyde (SSA), which is then oxidized to succinate by succinic semialdehyde dehydrogenase (SSADH).Thus, the carbon skeleton of GABA enters the TCA cycle (Wu et al., 2006).Among the three enzymes, GAD, which catalyzes the irreversible decarboxylation of glutamate in the presence of the cofactor pyridoxal 5'-phosphate (PLP), is considered to be the key enzyme for the biosynthesis of GABA (Huang et al., 1990).Data from the rice genome project show that there exist five GAD genes in rice (Project IRGS 2005).In the past 10 years, OsGAD1 and OsGAD2 have been intensively investigated (Akama et al., 2009;Akama et al., 2001;Akama et al., 2007).However, little is known about other family members such as OsGAD3.
Here we report on the cloning and characterization of the OsGAD3 gene from the GABA-rich giant-embryo of rice (Shangshi jing 315, SSJ315).Homologous alignment was performed to characterize the catalytic function of OsGAD3 (SSJ315).Structural feature analysis of the C-terminal portions of GADs and the evolution profiles of OsGAD3 (SSJ315) were also investigated.This work should provide further understanding of the important step involved in GABA biosynthesis in the GABA-rich giant-embryo rice at the molecular level.

Materials
Seeds of O. sativa (SSJ315), which were the GABArich giant-embryo rice and the hybrid offspring from "Haiminori" and "Xiushui 110", obtained from Prof. Dong (Shanghai Normal University), were grown in pots in the greenhouse of our laboratory under 30°C with 12 h light period (white fluorescent tubes: irradiance of 350 μmol m −2 s −1 ) and relative air humidity of 60%.The pMD-18T vector and one Step RNA PCR Kit was purchased from TaKaRa Biotechnology Co., Ltd.Primers' synthesis and DNA sequencing was performed by the Shanghai Sangon Biotechnological Company, China.All the other chemicals used were of analytical grade.

RNA extraction
The leaves (0.3 g) were excised from two-week-old O. sativa (SSJ315), pulverized in liquid nitrogen with mortar and pestle, and the total cellular RNA was extracted using the method reported previously.The quality and concentration of the extracted RNA were checked and stored as previously described (Kai et al., 2006).The RNA samples were stored at -80 o C until use.

Isolation of the full-length cDNA of GAD3 from
Oryza sativa (SSJ315) The cDNA was synthesized using the TaKaRa RNA PCR Kit (TaKaRa, Japan).Using cDNA as a template, RT-PCR was carried out in order to obtain OsGAD3.The reverse transcription reactions were performed by the sequential addition of the following reagents: 50 ng cDNA template, 25 mM MgCl2, 1 μl 10×RT buffer, 10 mM dNTPs, 0.25 μl RNase inhibitor, 0.5 μl AMV reverse transcriptase and 0.5 μl primers.The amplified product was purified and cloned into a pMD-18T vector and transformed into E. coli DH5a.Based on the color reaction using the Xgal-IPTG System and PCR identification, the positive clones were picked out and sequenced by ABI 3730 Sequencer (Perkin-Elmer, USA).

Bioinformatics analysis
Several websites and bioinformatics softwares were used for the analyses of OsGAD3.The nucleotide sequence, deduced amino acid sequence and open reading frame (ORF) were analyzed, and the sequence comparison was conducted through a database search using the BLAST program (NCBI, National Center for Biotechnology Services, http://www.ncbi.nlm.nih.gov).SOPMA analyses were performed online (http://www.expasy.org).
OsGAD3 and other GADs retrieved from GenBank were aligned with CLUSTAL W using default parameters.A phylogenetic tree was constructed using MEGA 2 (Kumar et al., 2001) combined with CLUSTAL W alignments.

Cloning and sequencing of the ORF of OsGAD3 (SSJ315)
The OsGAD3 was cloned using the primers that were designed according to the OsGAD3 (O.sativa japonica cultivar-group) sequence (AY187941).The ORF of OsGAD3 was 1479 bp and shared a homology with OsGAD3 (AY187941) of 99% with BLAST Search in GenBank database (http://www.ncbi.nih.gov).

Bioinformatics analysis
The OsGAD3 from O. sativa (SSJ315) contains a 1479 bp ORF encoding a protein of 492 amino acid residues with a calculated molecular mass of 56.1 kDa and a calculated isoelectric point (pI) of 5.72, which is similar to other plant GADs (Akama et al., 2001;Liu et al., 2004).
It is well known that a CaM-binding domain (CaMBD) exists in the C-terminal region of most plant GADs (Akama et al., 2001;Akama et al., 2007;Baum et al., 1993).In order to identify whether OsGAD3 (SSJ315) also contains the typical CaMBD or not, the C-terminal amino acid sequences (the last 32 amino acids) from various plant GADs were compared.As shown in Fig. 3, a Trp (W) residue at the center of this region and a lysine (K) cluster at the C-proximal position (indicated with a box in Fig. 3), which contribute to hydrophobic and electrostatic interactions respectively, were also found in OsGAD3 (SSJ315).These results reveal that the typical features in OsGAD3 (SSJ315) are conserved in all the compared plant GAD isoforms.
To investigate the evolutionary relationship of GADs, the phylogenetic tree was constructed based on the deduced amino acid sequences of OsGAD3 (SSJ315) and other GADs from different organisms including plants, bacteria and animals.The result revealed that GADs are derived from a common ancestor that evolved into three groups; OsGAD3 (SSJ315) belongs to the plant group and has the closest relationship with the GAD from Z. mays, which also belongs to the species of monocotyledonous plants as O. sativa (Fig. 4).This suggests that plant GADs are conserved in the process of evolution and GADs from the same species (like monocotyledonous plants) have a closer relationship in the process of evolution.

CONCLUSION
The molecular cloning and characterization of OsGAD3 (SSJ315) showed that it was very similar to other glutamate decarboxylases, indicating that OsGAD3 (SSJ315) belongs to a glutamate decarbo-The completely identical, highly similar and similar amino acids are indicated with "*", ":" and "." respectively.The black boxes indicate identical residues; the grey boxes indicate identical residues for at least three of the sequences.xylase superfamily.OsGAD3 (SSJ315) also possesses the typical CaMBD in the C-terminal region like most other plant GADs.Evolution analysis revealed that OsGAD3 (SSJ315) belongs to the plant group and plant GADs are conserved in the process of evolution.This work should be useful to further understand the important step involved in GABA biosynthesis in GABA-rich giant-embryo rice at the molecular level in the near future.