Department of Plant Pathology and Crop Physiology, College of Agriculture, Louisiana State University,. Baton Rouge, Louisiana 70803-1720. ABSTRACT.
Received for publication April 30, 1991 Accepted July 17, 1991
Plant Physiol. (1991) 97, 832-835 0032-0889/91 /97/0832/04/$01 .00/0
Communication
Hygromycin Resistance Gene Cassettes for Vector Construction and Selection of Transformed Rice Protoplasts' Zhenwei Zheng, Akio Hayashimoto2, Zhijian Li, and Norimoto Murai* Department of Plant Pathology and Crop Physiology, College of Agriculture, Louisiana State University, Baton Rouge, Louisiana 70803-1720 tridge plasmids containing plant-expressible hygromycin resistance genes.
ABSTRACT Hygromycin resistance gene cassettes were designed to facilitate vector construction for plant transformation. Unique EcoRI, Pstl, and Sacil sites in the coding sequence of a hygromycin B phosphotransferase gene (hph) from Escherichia coil were eliminated. The mutated hph genes were used to form gene cassettes
MATERIALS AND METHODS
Chemicals and Enzymes Restriction enzymes, T4 DNA polynucleotide kinase, Klenow fragment, T4 DNA polymerase, and Sequenase were purchased from Biolab, Bethesda Research Laboratories, or United States Biochemical and were used as specified by suppliers. Cellulase RS and Macerozyme R- 10 were obtained from Yakult Honsha (Tokyo, Japan). SeaPlaque agarose was acquired from FMC Co. Hygromycin B was obtained from Sigma Co.
flanked by EcoRI-Sacil-Kpnl-Hindlll sites. Hygromycin resistance of wild-type and mutated hph genes was indistinguishable in E. coli and rice protoplast growth assay.
Hygromycin B is an aminocyclitol antibiotic produced by Streptomyces hygroscopicus (1 1). It inhibits protein synthesis in both prokaryotes and eukaryotes, interfering with ribosomal translocation and aminoacyl-tRNA recognition (1, 3) and causing misreading of mRNA (14). A plasmid-encoded hygromycin resistance gene in Escherichia coli was isolated and sequenced by Gritz and Davies (4). The gene is 1026 base pairs long, encoding for hygromycin phosphotransferase with a predicted mol wt of 39,000. The enzyme phosphorylates hygromycin B and detoxifies it. A native gene was successfully used as a selectable marker for transformation of E. coli and Saccharomyces cerevisiae (4). Chimeric hph3 genes were used also for transformation of mammalian (10), tobacco (2), and rice cells (5). We are studying developmental control of rice storage protein glutelin. As a first step toward dissecting transcriptional regulatory elements, we have constructed a series of 5'upstream deletion mutations of the glutelin Gt- 1 gene (9). To facilitate construction of rice transformation vectors in pUC and pBluescript phagemids, we designed hygromycin resistance gene cassettes. Here, we report elimination of unique EcoRI, PstI, and SacII sites from the hph-coding sequence by oligonucleotide-directed mutagenesis and construction of car-
Hygromycin Resistance Gene Cassettes
The wild-type hph-coding sequence in pLG90 was kindly provided by Dr. Linda Gritz, Applied Biotechnology, Cambridge, MA (4). It was isolated as a 1. 1-kilobase pair fragment and cloned into a plant expression vector pTRA 105, resulting in pTRA132 (5). pTRA105 contained the 35S promoter of CaMV and transcription terminator originated from a tumor morphology large gene (tml) in transferred-DNA of Agrobacterium tumefaciens pTi 1 5955. General procedures for DNA manipulations were described by Sambrook et al. (12). Oligonucleotide-directed mutagenesis was carried out using the method of Kunkel et al. (6) to eliminate unique EcoRI, PstI, and SacII sites from the hphcoding sequence. The coding region was cloned into a BamHI site of a phagemid pBluscript KS(-) (Stratagene). The resulting plasmid was transformed to E. coli strain CJ236 (dut-, ung-) (Bio-Rad), and uracil-containing single-stranded DNA (minus strand) was prepared after superinfection of cells with a helper phage Ml 3K07 (Bio-Rad). Oligonucleotides were synthesized by Genetic Designs Co. (Houston, TX) or Oligos Etc. Inc. (South Orange, NJ). Oligonucleotide hph- 1 was an EcoRI site remover: 5'-CTCGCTAAACTCCCC-3' (the altered bases are underlined); hph-2 was a PstI site remover: 5'GACCGGTTGTAGAAC-3'; and hph-3 was a SacII site remover: 5'-ACGCCCGGAGTCGTG GCGAT-3'. First, we eliminated EcoRI and PstI sites, and the mutated hph was placed back in a shuttle vector pEX-2 (4) to test hygromycin
'This work was supported in part by grants from Louisiana Education Quality Support Funds (1987-90)-RD-A-6 and from Ishihara Sangyo Kaisha to N.M. 2 Present address: Department of Biotechnology, Ishihara Sangyo Kaisha, Ltd., Kusatsu, Shiga 525, Japan. 3Abbreviations: hph, hygromycin B phosphotransferase gene, CaMV, cauliflower mosaic virus. 832
HYGROMYCIN RESISTANCE GENE CASSETTES
5'-AATT CCGCGG TACC AAGCTT GGTA CCGCGG-3'
HP
HindlIl
KpnI
Figure 1. Nucleotide nucleotides.
sequence
Kpnl
EP
B
ESCI
SacIl
P
S
B
HP
B
S
0.3kb
ESKH P
pTRA151
B
P
M 0.3kb
B
35S
hph
_
EHKSE tml
The procedures for preparation and PEG-mediated transformation of protoplasts from the rice (Oryza sativa L.) cv Nipponbare were described by us previously (5, 7, 8). Selection for hygromycin-resistant transformants was applied on 14-dold regenerated protoplast calli for 12 d in general protoplast medium containing 95, 190, or 285 AM hygromycin B. Agarose blocks (10 x 10 x 0.7 mm) were transferred to soft agarose General medium containing 0.25% (w/v) Sigma type I agarose and 3% (w/v) sucrose. The number of hygromycinresistant colonies was counted in at least 10 agarose blocks after 2 weeks of propagation in soft agarose culture. The apparent transformation frequency of protoplasts was defined as the number of resistant calli per million protoplasts originally plated in agarose blocks.
Hygromycin B Concentration (MM) Incubation (h)
hph Gene
475
190
95 8
4
-a
_
_
++ ++ ++
+++ +++ +++
+ + ++
950
4 8 12 relative growth (A6w)
8
12
12
±
+ ±
±
+
+++ ++ +++
±
+
++ ++ ++
+++ ++ +++
Sacll-) _, no growth;
B
1.1kb
Table I. Growth of E. coli HB101 Containing Wild-Type or Mutated hph Genes in Different Concentrations of Hygromycin B An hph-coding sequence was inserted into the BamHl site of a shuttle vector pEX2 (3). The density of E. coli in L broth was determined by measuring absorbance at 600 nm.
a
EHKSE
Hygromycin B Resistance in Rice Protoplasts
E. coli strains were grown in L broth or on plates containing the same medium plus 1.5% agar. Four concentrations of hygromycin B (95, 190, 475, and 950 ,uM) were prepared to select resistant colonies on plates or in liquid. Overnight culture (1 mL) developed from a single colony was inoculated into 50 mL of L broth and incubated at 37°C with shaking (250 rpm). The absorbance was measured at 600 nm at 30min intervals.
None Wild-type Mutated (EcoRl, Pstl-) Mutated (EcoRlI, Pstl-,
-
Figure 2. Physical organization of wild-type and mutated hygromycin resistance gene cassettes in pUC4 vectors. Plasmid pTRA132 contains the wild-type hph-coding sequence; pTRA1 41 and pTRA1 50 contain the mutated hph-coding sequences without EcoRl and Pstl sites; pTRA1 51 contains the mutated hph-coding sequence without EcoRl, Pstl, and SaclI sites. B, BamHl; E, EcoRl; H, Hindlil; K, Kpnl; P, Pstl; S, SaclI; kb, kilobase; 35S, 35S promoter of CaMV; tml, transcription terminator of a tumor morphology large gene.
Hygromycin B Resistance in E. coli
4
E M
B
-1i
pTRA150
resistance in the E. coli stain HB 101. The mutated hph-coding sequence (EcoRI-, Pstl-) was isolated from one resistant colony and cloned in the plant expression vector pTRA 105. The EcoRI site of the resultant plasmid was replaced by the HindIll or PstI site, forming a HindIlI or PstI hygromycin resistance gene cassette (the CaMV 35S promoter-hph coding sequence-tml terminator), respectively. The PstI gene cassette is in pTRA 150 as shown in Figure 2. HindIII-EcoRI-SacIIKpnI oligonucleotides were annealed to form double-stranded DNA fragments containing SacII-KpnI-HindIII-KpnI-SacII restriction sites and EcoRI overhangs (Fig. 1). The annealed linker was introduced in pUC4-KSAC (Pharmacia) at the EcoRI site, forming pUC4-KESH (the kanamycin resistance gene was deleted). The HindIlI cassette was inserted into the HindIII site of the pUC4-KESH vector, forming pTRA 141 as shown in Figure 2. Later, the SacII site was also removed from the hph-coding sequence, and the newly mutated hph (EcoRI-, PstlP, and SacII-) was used to form a KESH hygromycin resistance gene cassette in pTRA 151 (Fig. 2). These introduced mutations were confirmed by DNA sequencing (13).
B
1_'
pTRA141
of Hindlil-EcoRl-Sacill-Kpnl oligo-
S
I I
pTRA132
3'-GGCGCC ATGG TTCGAA CCAT GGCGCC 'I IAA-5', SaclI
833
±, A60Q < 0.5; +, A600 = 0.5 to 1.5; ++, A600 = 1.5 to 2.5; +++, A600 > 2.5
+
±
ZHENG ET AL.
834
Plant Physiol. Vol. 97, 1991
Table Il. Apparent Transformation Frequency of Rice (cv Nipponbare) Protoplasts by Wild-Type (pTRA 132) or Mutated Hygromycin Resistance Genes (pTRA 141, pTRA 150, or pTRA 151) Numbers of hygromycin-resistant calli per million treated rice protoplasts are listed. Each value was an average of two independent experiments. SE values are in parentheses. Apparent Transformation Frequency Hygromycin B concentration (uM)
hph Genes
285
190
95
no. resistant calli per million
None pTRA132 (wild-type) pTRA141 (EcoRl-, Pstl-) pTRA150 (EcoRlI, Pstl-) pTRA151 (EcoRlI, Pstl-, Sacll )
0 248.6 (25.8) 367.0 (159.7) 288.8 (69.9) 270.4 (28.6)
RESULTS AND DISCUSSION Construction of Hygromycin Resistance Gene Cassettes
We designed hygromycin resistance gene cassettes to facilitate introduction of a hygromycin resistance marker into rice transformation vectors. Unique EcoRI, PstI, and SacIl sites in the hph-coding sequence were eliminated by oligonucleotide-directed mutagenesis without altering the corresponding amino acid sequence. Thus, a hygromycin resistance gene cassette (1.7 kilobases) can be easily generated from the cassette vectors after digestion with either EcoRI, PstI, HindIII, KpnI, or SaclI restriction endonuclease (Fig. 2). DNA sequence analysis of the mutated hph in pTRA 151 demonstrated that the EcoRI, Pstl, and SacIl recognition sequences were eliminated after modifying GAATTC (altered bases are underlined) to GAGTTT, CTGCAG to CTACAA, and CCGCGG to CCACGA, respectively. We found that the last codon was AAA (Lys) and not GAA (Glu) as reported by Gritz and Davies (4). The hygromycin resistance gene cassettes can be modified further by eliminating additional restriction sites for HinclI, HphI, NcoI, NdeI, and PvuI in the hph-coding region or by flanking the cassettes with different restriction sites.
Expression of Hygromycin B Resistance in E. coli Wild-type and mutated hph-coding sequences were placed into a shuttle vector pEX-2 (4), under control of the cyc 1 promoter. Both wild-type and mutated hph genes conferred to E. coli strains HB 101, XL 1-Blue, and K802 resistance up to 950 ,iM of hygromycin B on plates or in liquid medium as summarized in Table I for E. coli HB 101. None of these strains in the absence of a hygromycin resistance gene could grow in a hygromycin concentration >95 ,uM. In general, the higher the concentration of hygromycin was, the slower the growth of E. coli. Transformation frequency of E. coli cells was essentially equivalent at the 50 ,ug/mL concentration of ampicillin or hygromycin B (data not included). Interestingly, E. coli cells containing the hph cassette vectors pTRA 141, pTRA 1 50, and pTRA 1 51 were resistant to hygromycin B. A mechanism of expression of this hygromycin
0 233.5 (5.7) 298.8 (163.2) 257.3 (150.3) 221.1 (28.9)
0 239.5 (10.6) 199.0 (72.3) 248.6 (121.5) 201.4 (21.4)
resistance in E. coli is not known. It may be readthrough from other prokaryotic promoters in pUC4. However, hygromycin resistance of the cassette vectors in E. coli was orientation independent in reference to the direction of transcription of the f-lactamase, lac Z, and CaMV 35S promoters. Hygromycin B Resistance in Transformed Rice Protoplasts
Transformation of both prokaryotic and eukaryotic cells relies heavily on the use of dominant resistance phenotypes as selectable markers. Because hygromycin B is a potent inhibitor of protein synthesis in both prokaryotes and eukaryotes, the hph genes have been particularly effective in stringent selection of transformed rice cells (5). Fertile transgenic rice plants were regenerated from hygromycin-resistant calli. We compared the apparent transformation frequency of rice protoplasts after treatment with wild-type (pTRA 132) or one of the mutated hph genes (pTRA 141, pTRA 150, or pTRA 151). Three concentrations of hygromycin B (95, 190, or 285 ,uM) were applied to 14-d-old regenerated protoplasts, and hygromycin selection was continued for 2 weeks. There were no statistical differences in the number of resistant calli after transformation with either pTRA 132, pTRA 141, pTRA 1 50, or pTRA 151 (Table II). Both wild-type and mutated hph genes conferred resistance to rice cells up to 285 AM of hygromycin B. We conclude that elimination of EcoRI, PstI, and SacIl sites from the wild-type hph-coding sequence did not affect the level of hygromycin resistance phenotype of the hph gene. The hygromycin resistance gene cassettes that were constructed here should be extremely useful for selection of other higher plant cells. These gene cassette plasmids will be available upon request. ACKNOWLEDGMENTS We sincerely thank Ms. Linda Gritz for providing pLG90 Drs. Mary E. Musgrave and Alan J. Biel for critical reading of the manuscript, and all members of the Plant Molecular Biology Laboratory for discussion and encouragement.
HYGROMYCIN RESISTANCE GENE CASSETTES LITERATURE CITED 1. Cabanas MJ, Vazquez D, Modolell T (1978) Dual interference of hygromycin B with ribosomal translocation and with aminoacyl-tRNA recognition. Eur J Biochem 87: 21-27 2. Elzen PJM van den, Townsend J, Lee KY, Bedbrook JR (1985) A chimeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol Biol 5: 299-302 3. Gonzalez A, Jimenez A, Vazquez D, Davis J, Schindler D (1978) Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochim Biophys Acta 521: 459-469 4. Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25: 179-188 5. Hayashimoto A, Li Z, Murai N (1990) A polyethylene glycolmediated protoplast transformation system for production of fertile transgenic rice plants. Plant Physiol 93: 857-863 6. Kunkel TA, Roborts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154: 367-382 7. Li Z, Murai N (1990) Efficient plant regeneration from rice protoplasts in general medium. Plant Cell Rep 9: 216-220
835
8. Li Z, Burow MD, Murai N (1990) High frequency generation of fertile transgenic rice plants after PEG-mediated protoplast transformation. Plant Mol Biol Rep 8: 276-291 9. Okita TW, Hwang YS, Hnilo J, Kim WT, Aryan AP, Larson R, Krishnan HB (1989) Structure and expression of the rice glutelin multigene family. J Biol Chem 264: 12573-12581 10. Palmer TD, Hock RA, Osborne WRA, Miller AD (1978) Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine-deficient human. Proc Natl Acad Sci USA 75: 1055-1059 11. Pettinger RC, Wolfe RN, Hoehn MM, Marks PN, Dailey WA, McGuire JM (1953) Hygromycin. I. Preliminary studies on the production and biological activity of a new antibiotic. Antibiotics Chemother 3: 1268-1278 12. Sambrook J, Fritsch F, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York 13. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 14. Singh A, Ursic D, Davies J (1979) Phenotypic suppression and misreading in Saccharomyces cerevisiae. Nature 277: 146-148
