Biosci. Biotechnol. Biochem., 72 (11), 3041–3043, 2008
Note
Construction of a Binary Vector for Transformation of Arabidopsis thaliana with a New Selection Marker Nozomu K OIZUMIy and Yuji I WATA* Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan Received June 25, 2008; Accepted July 23, 2008; Online Publication, November 7, 2008 [doi:10.1271/bbb.80428]
A new binary vector, pZT4B, containing the UDP-Nacetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as a selection marker, was constructed. The green fluorescent protein (GFP) gene was inserted into pZT4B, and the resulting plasmid was used in the transformation of Arabidopsis. All of six independent transformants obtained after selection with 0.3 mg/l tunicamycin contained the transgene and showed GFP fluorescence. Key words:
Arabidopsis; binary marker; tunicamycin
vector;
selection
Transgenic plants are routinely generated in the laboratory for both basic and applied research purposes. Two approaches can be used to transform plants: direct gene transfer and Agrobacterium tumefaciens-mediated gene transfer.1,2) In most cases, one or more selection marker genes are co-integrated with the genes of interest in order to identify plants with the desired genes in their genome. In general, selection marker genes encode enzymes that detoxify antibiotics that are toxic to plants. Genes encoding neomycin phosphotransferase II3,4) and hygromycin phosphotransferase,5) which detoxify kanamycin and hygromycin respectively, are widely used in the transformation of plants. Genes encoding enzymes that detoxify herbicides are also used.6) It is often necessary to introduce multiple genes into a plant genome. For this purpose, different selection markers are necessary, because transgenic plants already contain one or more selection marker genes. In Arabidopsis, for example, T-DNA insertion mutants are powerful tools to investigate the functions of genes.7) Most T-DNA insertion mutants contain at least one selection marker gene, and an additional selection marker gene is then necessary to re-introduce the genes. In many cases, it is possible to deal with this by using currently available multiple selection markers.8) Howy
ever, it would be advantageous for researchers if more selection markers that are convenient for use were available. Previously we found that the GPT gene could be used as a selection marker when it is overexpressed in Arabidopsis grown in the presence of tunicamycin.9) Present study was conducted to construct a new binary plasmid using this selection system. The starting plasmid, pPZP2028 (Kuroda et al., unpublished) was constructed by modification of pPZP202:10) two eight-base-cutter restriction sites, Asc I and Pac I, were introduced in accordance with the method of van Engelen et al.11) A gene cassette comprising the cauliflower mosaic virus 35S (CaMV35S) promoter, GPT cDNA, and the nopaline synthase (Nos) terminator was amplified by PCR from a binary plasmid described previously,12) using primers containing the Mlu I site on the 50 end and the Asc I site on the 30 end. The PCR product was digested with Mlu I and Asc I, then ligated with pPZP2028 that had been digested with Asc I, and treated with phosphatase, yielding pZT4B. Subsequently, another gene cassette, comprising the CaMV35S promoter, sGFP and the Nos terminator, was amplified by PCR from CaMV35S-sGFP(S65T)NOS30 13) using primers with the Xho I or EcoR I site, and digested with Xho I and EcoR I. The resulting fragment was ligated with pZT4B that had been digested with Sal I and EcoR I to obtain pZT4B-GFP. The method used to construct the vectors is summarized in Fig. 1. pZT4B-GFP was used for in planta A. tumefaciensmediated transformation of Arabidopsis (ecotype Columbia) in accordance with the method of Clough and Bent.14) T0 seeds were sown on agar plates (1 Murashige and Skoog salt, 1 Gamborg’s B5 vitamins, 1% w/v sucrose, and 0.8% w/v agar, pH 5.6) supplemented with 0.3 mg/l tunicamycin, and incubated at 22 C. Most seedlings died just after germination, but some continued to grow and developed true leaves.
To whom correspondence should be addressed. Present address: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan; Tel/Fax: +81-72-254-9424; E-mail:
[email protected] * Present address: Huck Institutes of the Life Sciences, Pennsylvania State University, 211 Wartik Laboratory, University Park, PA 16802, USA Abbreviations: CaMV35S, cauliflower mosaic virus 35S; GPT, UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase; Nos, nopaline synthase; sGFP, synthetic green fluorescent protein
3042
N. KOIZUMI and Y. IWATA
pPZP2028
1
SpecR LB
Asc I
MCS
Pac I
RB
2 3
4
5 6 7
GFP
0.7 kbp
GPT
0.9 kbp 0.6 kbp
Hind III, Sph I, Pst I, Sal I, BamH I, Spe I, Kpn I, Sac I, EcoR I Mlu I 35S-P
GPT
Nos-T
Asc I Asc I
pZT4B SpecR Asc I
LB Nos-T
GPT
35S-P
35S-P
GFP
Nos-T
Pac I
MCS
RB
Xho I Sal I/EcoRI EcoRI
pZT4B-GFP SpecR Asc I
LB Nos-T
GPT
35S-P
35S-P
Pac I GFP
RB
Nos-T
Fig. 1. Strategy Used in Plasmid Construction. SpecR, LB, and RB represent the spectinomycin resistant gene, left border and right border respectively. Mlu I and Asc I generate complementary cohesive ends. Similarly, cohesive ends generated by Xho I and Sal I are ligatable. A Sph I site exists on the right border of pPZP2028 and a Hind III site exists in the GPT sequence. Hence the Sph I and Hind III sites in the multicloning site (MCS) are not available.
These seedlings were transferred to soil and grown to harvest the seeds. Seeds from six independent T1 plants were sown on plates containing tunicamycin (0.3 mg/l), and the resulting T2 seedlings from all lines showed resistance to tunicamycin at different segregation rates (data not shown), suggesting that all six T1 plants contained the transgene. To confirm the transformation event, genomic DNA was isolated from these six lines and subjected to PCR using primers amplifying the GPT genes. As shown in Fig. 2, a signal for the authentic GPT gene and an extra signal derived from the transgene were detected in all transgenic lines, but not in the wild-type plants. In addition, all six lines showed clear fluorescence under fluorescent microscopy (data not shown). These observations confirmed that all lines were transformants. Seeds from wild-type and transgenic plants (one line only) were sown on plates with and without tunicamycin (0.3 mg/l). The GFP fluorescence of these seedlings was monitored. As shown in Fig. 3, wild-type plants barely grew on plates with 0.3 mg/l tunicamycin, and did not show any fluorescence. In contrast, transgenic plants grew in the presence of tunicamycin and showed clear fluorescence. Not all transgenic seedlings fluoresced, because this was a T1 line that showed segregation. These results indicate that pZT4B can be used for in planta transformation of Arabidopsis.
Fig. 2. Detection of Transgenes. Genomic DNA was isolated from T2 seedlings using a Nucleon Phytopure DNA extraction kit (GE Healthcare UK, Buckinghamshire, UK). Detection of GFP and GPT genes was carried out by PCR using following primers: 50 -TCTAGAATGGTGAGCAAGGGCGAGGAG-30 and 50 -GGATCCCTTGTACAGCTCGTCCATGCC30 for GFP, and 50 -TCTTCTCTGTTCGCTCTATCTATACC-30 and 50 -GTTTGTACAAAAGACAGCAAGTAGCC-30 for GPT. Amplified products were fractionated by agarose gel electrophoresis. Lane 1, wild-type plants; lanes 2 to 7, independent transgenic plants. The size of the GFP fragment was 729 bp. The size of the fragment derived from the endogenous GPT gene was 925 bp, and the predicted size of the fragment derived from the transgene was predicted to be 562 bp.
Bright
GFP
-Tm
+Tm
Fig. 3. Tunicamycin Resistance and GFP Fluorescence of Transgenic Plants. Wild-type and transgenic Arabidopsis seedlings were grown on plates with (+Tm) and without (Tm) tunicamycin (0.3 mg/l) on a 16-h light/8-h dark cycle at 22 C, and 6-d-old seedlings were photographed. The presence of GFP fluorescence was assessed in the same seedlings using an LAS-1000plus CCD camera (Fujifilm, Tokyo). Seeds of wild-type (upper half of each plate) and transgenic plants (lower half of each plate) were sown on plates with (C, D) and without (A, B) tunicamycin. Photos were taken under normal light (A, C) and fluorescent light (B, D).
The selection procedure is basically the same as the method used with other drugs, such as kanamycin and hygromycin, but clearer selection is possible, because selection by tunicamycin is quite distinctive, as described previously.9)
A New Binary Vector for Arabidopsis
In the present study we generated a new binary vector pZT4B that contains the GPT gene as a selection marker. This vector was confirmed to function at least in Arabidopsis, and should be useful to Arabidopsis researchers. Since pZT4B contains two eight-base-cutter restriction sites, Asc I and Pac I, introduction of multiple genes into pZT4B can easily be achieved (Kuroda, personal communication). The CaMV35S promoter driving the GPT gene might affect the expression of desired genes cloned into the multicloning site, since the CaMV35S promoter was recently reported to affect the expression of adjacent genes.15) Hence another promoter such as Nos promoter may be more suitable for driving the GPT gene but it is currently unclear whether the Nos promoter is sufficiently strong to overexpress the GPT gene to achieve tunicamycin resistance. Use of the plant gene as a selection marker might be advantageous if this system is used in the generation of transgenic crops, since the Food and Agriculture Organization of the United Nations (FAO) has recommended that selection markers derived from bacteria not be used. Another endogenous gene of Arabidopsis has also been reported to be effective as a selection marker.16) Application of this system to crop plants is the next challenge.
Acknowledgments We thank Dr. Yasuo Niwa (Shizuoka Prefecture University) for CaMV35S-sGFP(S65T)-NOS30 and Dr. Masaharu Kuroda (National Agricultural Research Center) for pPZP2028. We also appreciate the excellent technical assistance of Ms. Naoko Yamaguchi of our laboratory. This work was partly supported by a Grantin-Aid for Scientific Research (grant no 20389188) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Feasibility Study of Japan Science, and Technology Agency.
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
References 1)
2)
3)
Klein, T. M., Harper, C. E., Svab, Z., Sanford, J. C., Fromm, M. E., and Maliga, P., Stable genetic transformation of intact Nicotiana cells by the particle bombardment process. Proc. Natl. Acad. Sci. USA, 85, 8502–8505 (1988). Bevan, M., Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res., 12, 8711–8721 (1984). Herrera Estrella, L., De Block, M., Messens, E., Hernalsteens, J. P., Van Montagu, M., and Schell, J.,
15)
16)
3043
Chimeric genes as dominant selectable markers in plant cells. EMBO J., 2, 987–995 (1983). Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P. R., Flick, J. S., Adams, D. P., Bittner, M. L., Brand, L. A., Fink, C. L., Fry, J. S., Galluppi, G. R., Goldberg, S. B., Hoffmann, N. L., and Woo, S. C., Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. USA, 80, 4803–4807 (1983). Waldron, C., Murphy, E. B., Roberts, J. L., Gustafson, G. D., Armour, S. L., and Malcolm, S. K., Resistance to hygromycin-B: a new marker for plant transformaion studies. Plant Mol. Biol., 5, 103–108 (1985). Li, Z., Hayashimoto, A., and Murai, N., A sulfonylurea herbicide resistance gene from Arabidopsis thaliana as a new selectable marker for production of fertile transgenic rice plants. Plant Physiol., 100, 662–668 (1992). Azpiroz-Leehan, R., and Feldmann, K. A., T-DNA insertion mutagenesis in Arabidopsis: going back and forth. Trends Genet., 4, 152–156 (1997). Miki, B., and McHugh, S., Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J. Biotechnol., 107, 193–232 (2004). Koizumi, N., A chimeric tunicamycin resistance gene as a new selectable marker for Arabidopsis thaliana. Plant Biotech., 20, 305–309 (2003). Hajdukiewicz, P., Svab, Z., and Maliga, P., The small, versatile pPZP family of Agrobacterium binary vectors for plants. Plant Mol. Biol., 25, 989–994 (1994). van Engelen, F. A., Molthoff, J. W., Conner, A. J., Nap, J.-P., Pereira, A., and Stiekema, W. J., pBINPLUS: an improved plant transformation vector based on pBIN19. Trans. Res., 4, 288–290 (1995). Koizumi, N., Ujino, T., Sano, H., and Chrispeels, M. J., Overexpression of a gene that encodes the first enzyme in the biosynthesis of Asn-linked glycans makes plants resistant to tunicamycin and obviates the tunicamycininduced unfolded protein response. Plant Physiol., 121, 353–362 (1999). Sheen, J., Hwang, S. B., Niwa, Y., Kobayashi, H., and Galbraith, D. W., Green-fluorescent protein as a new vital marker in plant cells. Plant J., 8, 777–784 (1995). Clough, S. J., and Bent, A. F., Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J., 16, 735–743 (1998). Zheng, X., Deng, W., Luo, K., Duan, H., Chen, Y., McAvoy, R., Song, S., Pei, Y., and Li, Y., The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep., 26, 1195–1203 (2007). Mentewab, A., and Stewart, C. N. Jr., Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants. Nat. Biotechnol., 23, 1177–1180 (2005).
