Os03g0264400

OASA2—an a-subunit of anthranilate synthase that is a key enzyme of tryptophan (Trp)vbiosynthesis in rice (Oryza sativa)

Annotated Information

Function

• OASA2—an a-subunit of anthranilate synthase that is a key enzyme of tryptophan (Trp)vbiosynthesis in rice (Oryza sativa)

Expression

• The expression level of OASA2 in plants homozygous and heterozygous for modified OASA2 was similar to that of nontransformants, suggesting that OASA2 transcription in GT plants was controlled in the same manner as endogenous OASA2, and that GTcould lead to a lower risk of gene silencing than in conventional overexpression approaches.One potential problem for selecting GT products was that transcript levels of mutated OASA2 were expected to be relatively low in rice cells in which GT had occurred successfully because the mutated OASA2 is under the control of its own intrinsic promoter. Moreover, GT occurs at very low frequency compared to conventional transformation experiments.Thus, we optimized the concentration of 5MT required to select GT cells efficiently using a control vector containing a mutated OASA2 expression cassette driven by the intrinsic promoter and terminator (Fig. 2A); 500 mM 5MT was found to be optimal for selection of GT cells(Fig. 2B).

'Figure 2. GTexperiments to introduce mutations into OASA2. A, Schematic representation of GT vectors and the control vector used in this study. As GT vectors, a 7.0-kb fragment containing a partial OASA2 fragment with S126F/L530D or Y367A/L530D mutations and silent mutations for CAPS markers was constructed. As a control vector, a 7.5-kb fragment containing a full OASA2 expression cassette with mutations was constructed. The S126F, Y367A, and L530D mutations (S126F; S [TCC] to F [TTC] at amino acid 126, Y367A; Y [TAC] to D [GCC] at amino acid 367, L530D; L [CTT] to D [GAC] at amino acid 530) and silent mutations for the CAPS markers (added HindIII site at 16-bp upstream of S126F; CAGCTT to AAGCTT, deleted EcoRV site at 22-bp upstream of L530D; GATATC to GATTTC) were introduced into the OASA2 fragment. The Y367A mutation generates additional recognition sites for NcoI; ACATGG to CCATGG. Left, The green, white, and black boxes show the putative chloroplast-targeting signal, untranslated region, and coding region of OASA2 (Os03t0264400-01), respectively. Right, The gray and white boxes show the putative gene structure of Os03t0264300-00, respectively. The horizontal arrows show the direction of transcription of each gene. B, Selection of rice calli transformed with empty vector (pPZP2028, left section) and control vector(right section) and selected on medium containing 500 mM 5MT.

• The amount of free Trp in calli from both numbers 3-4 and 16-11 was significantly higher than in nontransformants (Fig. 3A), suggesting that GT events had occurred successfully in these lines.To allow sequencing of OASA2 in regenerated number 3-4 plant (T0 generation) and its progeny (T1 generation), PCR was performed with primers that anneal to the region outside the GT construct (not present on the GT vector). Direct sequencing of PCR fragments confirmed that GT had occurred successfully (Fig. 3B). Southern-blot analysis of T0 and T1 generations of number 3-4 using HindIII with probes 1 and 2 (Fig. 3C) showed that a true GT event in which the wild-type OASA2 locus was modified as expected had indeed occurred because the wild-type 6.0-kb band was not observed in lines 2 and 5 of the T1 generation.In addition, additional genomic rearrangements in the OASA2 locus were not observed in Southern-blot analysis using probes 1 and 3 (Fig. 3C). These results were confirmed by Southern-blot analysis using BamHI/EcoRV with probe 2 (Fig. 3D), which showed that a 6.6-kb band derived from randomly integrated T-DNA was successfully segregated out in lines 2 and 7 of the T1 generation (Fig. 3D).

'Figure 3. Molecular analysis of GT plants harboring OASA2 with S126F/L530D mutations. A,Bar charts showing free Trp content in calli of nontransformant (NT), GT line 3-4 in the T2 generation (heterozygous), and GT line 16-11 in the T0 generation. Values are average 6 SD (n =3). B, Sequencing chromatograms of the mutation site L530D of number 3-4 in T0 and T1 generations. C, Southern-blot analysis of HindIII-digested number 3-4 in T0 and T1 generations using probes 1 to 3 (purple bars). The wild-type (6.0 kb) and targeted (4.5 kb) bands were detected using probe 1.Wild-type (1.1 and 6.0 kb) and targeted band or bands derived from random integrated T-DNA(1.5 kb) were detected using probe 2. Wild-type bands (1.1 and 0.6 kb) were detected using probe 3. N, Nontransformant. D, Southern-blot analysis of BamHI/EcoRV-digested number 3-4. Wild-type(5.0 kb) and targeted (8.0 kb) bands were detected using probe 1. Wild-type bands (5.0 kb), a band derived from random integrated T-DNA (6.6 kb),and the targeted band (8.0 kb) were detected using probe 2. A wild-type band (3.5 kb) was detected using probe 3.'

Evolution

Please input evolution information here.

You can also add sub-section(s) at will.

Labs working on this gene

• Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8602, Japan (H.S., H.O., S.T.);
• RIKEN Plant Science Center,Yokohama, Kanagawa 230–0045, Japan (A.O., F.M., K.S.); Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263–8522, Japan (K.S.);
• Kihara Institute of Biological Research, Yokohama City University, Yokohama, Kanagawa 244–0813, Japan (S.T.)

References

• de Pater S, Neuteboom LW, Pinas JE, Hooykaas PJJ, van der Zaal BJ (2009) ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation. Plant Biotechnol J 7:821–835