Jun 28, 1988 - involved in that control. Acknowledgments-The authors are indebted to Marlaya Wyncott, Gary. Thompson, Patti Sherril, Mark Golub, Kelly Roth, ...
Plant Physiol. (1988) 88, 1002-1007
0032-0889/88/88/1002/06/$0 1.00/0
The Synthesis of a 19 Kilodalton Zein Protein in Transgenic Petunia Plants' Received for publication February 26, 1988 and in revised form June 28, 1988
JOHN D. WILLIAMSON, GAD GALILI2, BRIAN A. LARKINS, AND STANTON B. GELVIN* Department of Biological Sciences (J.D.W., S.B.G.) and Department ofBotany and Plant Pathology (G.G., B.A.L.), Purdue University, West Lafayette, Indiana 47907 ABSTRACT Transcriptional fusions composed of a 19 kilodalton zein cDNA, the 5' flanking region from a j-phaseolin gene, and 3' flanking regions from either the phaseolin or a 15-kilodalton zein gene were introduced into Petunia by Agrobacterium-mediated transformation. The expression of both zein mRNA and protein in these transgenic plants was seed-specific and developmentally regulated. Both monocot (zein) and dicot (phaseolin) polyadenylation consensus sequences were recognized in Petunia. Analysis by immunoblotting showed that the Mr of the zein protein corresponded to that of the mature protein, suggesting that recognition and cleavage of the signal sequence had occurred. While zein mRNA accumulated to approximately 1% of the total poly(A)' RNA in seeds of the transformed plants, zein protein was present at a much lower concentration than expected, at most being 0.005% of the total seed protein. These results suggest that the 19 kilodalton zein gene, in addition to lacking specific sequences required for efficient transcription in dicots, might also lack sequences required for the efficient synthesis, targeting, transport, or stabilization of the protein.
Zeins, the major storage proteins of maize seed, are of considerable agronomic importance, and as such have been the focus of much study. The organization, structure, and expression of genes encoding the various zeins have been characterized (6, 12, 36) as has the compartmentation of the proteins within the endosperm (19, 20). A structural model of the 19 and 22 kD azein proteins has also been proposed (2). Since the development of suitable vector systems for the Agrobacterium-mediated transfer of foreign DNA into plant tissue, there have been several studies on the expression of zein genes in undifferentiated sunflower tissue (11, 23, 27). These studies showed that zein genomic sequences contain sufficient information for their accurate transcription in sunflower tumors. However, the levels of zein mRNA were much lower than those found in developing maize endosperm, and zein proteins were not detected. The expression of seed storage protein genes in transgenic plants has recently been a topic of intense investigation. A gene encoding ,3-phaseolin, the major storage protein of French bean, directed the developmentally regulated accumulation of high levels of phaseolin mRNA and protein in tobacco seed (30). ' Supported by a grant from Agrigenetics Research Associates to S. B. G. G. G. was the recipient of a Chaim Weizmann postdoctoral fellowship from the Weizmann Institute of Science, Israel. 2 Present address: Department of Plant Genetics, Weizmann Institute of Science, Rehovot, 76100, Israel.
Similar results were obtained when a gene for an a' subunit of ,B-conglycinin, a major storage protein of soybean, was introduced into Petunia (3). In contrast, when a genomic sequence that encodes a 19 kD zein was introduced into Petunia, only low levels of zein mRNA were detected (35). In addition, the accumulation of zein mRNA was not seed-specific, nor was there detectable accumulation of the protein. Detectable amounts of 15 kD zein mRNA and protein were found in tobacco, however, when the 5' and 3' flanking sequences of a f3-phaseolin gene were used to direct expression of the 15 kD zein protein coding sequence (13). In contrast to its localization in maize endosperm, where it accumulates in ER-derived protein bodies (20), the 15 kD zein protein was found in large protein bodies within the embryo. The failure to detect the 19 kD zein protein in seeds of transgenic Petunia might be due to several factors, including the inability of dicots efficiently to recognize monocot transcriptional regulatory elements, the instability of the RNA, or the inability efficiently to translate, process, and store the protein. To examine these possibilities we constructed two chimeric 19 kD zein genes. In both cases, 5' flanking sequences from a 3lphaseolin gene were used to direct transcription in transgenic Petunia plants. In one construct, pSpA, the 3' flanking sequence from the ,3-phaseolin gene was used to direct polyadenylation, while in the other, pGZ322, the 3' flanking sequence from a 15 kD zein gene was used. Both of these constructs directed the seed-specific transcription of zein mRNA. The 19 kD zein protein was also synthesized, processed to the mature form and accumulated in developing seeds. However, the levels of zein protein were very low.
MATERIALS AND METHODS Plasmid Constructions. Phaseolin-zein chimeric genes were constructed as shown in Figure 1. For the construction of pSpA (Fig. 1 B), an 831 bp3 Hinfl fragment containing a 19 kD zein coding sequence was isolated from the cDNA clone cZ l 9cl (Fig. lA) (22). This fragment was treated with Klenow fragment of DNA polymerase I to produce blunt ends and inserted into the SmaI site of the binary plant transformation vector pBinl9 (4) (Fig. 1, step 1). The resulting plasmid was digested with BamHI, and an 850 bp BglII fragment containing the promoter and 5' untranslated region of the ,B-phaseolin genomic clone p8.8.pro (13) was inserted (Fig. 1, step 2). Finally, a 1.01 kbpAlul fragment containing the consensus polyadenylation signal sequence and 3' flanking region of p8.8.pro was inserted into the filled-in EcoRI site of the pBin 19 polylinker region (Fig. 1, step 3). The 3 Abbreviations: bp, base pair(s); dpp, days postpollination; MBN, mung bean nuclease; kbp, kilobase pair(s); SP6, Salmonella phage 6; RER, rough endoplasmic reticulum.
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SYNTHESIS OF ZEIN PROTEIN IN PETUNIA B
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FIG. 1. Construction of the phaseolin/zein chimeric genes. The plasmids and cloning steps (1 through 7) used in the construction of pSpA and pGZ322 are described in "Materials and Methods." Arrows indicate the direction of transcription. Sites separated by a (\) have been destroyed by the fusion of two different restriction sites. Sites marked with an (x) have been deleted. A, AluI; B, BamHI; Bg, BglII; E, EcoRI; H, HincII; H3, HindIII; Hf, Hinfl; K, KpnI; Pv, PvuII; Sm, SmaI; S, SstI; X, XbaI; Xh, XhoI.
resulting transcriptional fusion contains 750 nucleotides upstream of the phaseolin TATA consensus sequence, and 86 nucleotides between the phaseolin mRNA cap site and the 19 kD zein translation start site (ATG). This compares to approximately 80 nucleotides between the cap site and ATG in the ,Bphaseolin gene. The clone pGZ322 was constructed to allow excision and replacement of the zein coding sequence. This required the insertion of a SstI restriction endonuclease site between the phaseolin mRNA cap site and the zein translation start site. To maintain approximately equal spacing between the cap site and translation start sites in pSpA and pGZ322, 33 bp of the zein 5' untranslated sequence were deleted by Bal31 digestion as described by Galili et al. (9). The DNA sequence of the 5' end of the resulting plasmid, pSP6.cZ19cl.Awt, was determined by the method of Sanger et al. (28). The 850 bp BglII fragment containing the phaseolin promoter region from the plasmid p8.8.pro was inserted into the BamHI site of pUC 18 (Fig. 1A, step 4) from which the SstI site had been removed by digestion with MBN. The resulting clone was designated pUC1 8ASst.pro. The 828 bp EcoRI-Hinfl fragment containing the 5'-deleted zein coding sequence from plasmid pSP6.cZl9cl.Awt and a 315 bp Hinfl-XhoI fragment containing the polyadenylation consensus sequence from the 15 kD zein genomic clone pgZl5 (25) were inserted between the EcoRI and Sall restriction endonuclease sites of pUC1 8 (Fig. lA, step 5) to produce the clone p1551. The clone p1551 was digested with EcoRI and treated with MBN to produce blunt ends. The fragment containing the zein coding and polyadenylation sequences was released by digestion with HindIll. The resulting fragment, containing a blunt end and a HindIll end, was inserted between the XbaI site, which had been blunt-ended with MBN, and the HindIII site of plasmid pUC18ASst.pro (Fig. IA, step 6). The resulting plasmid was digested with HindIll and EcoRI, and the fragment containing the phaseolin-zein chimeric gene was inserted between the HindIll and EcoRI sites of the binary plant transformation vector pGA492 (1) (Fig. IA, step 7). Both plasmids carry a nos-nptll
fusion that confers kanamycin resistance upon plants. The recombinant plasmids were mobilized into A. tumefaciens strain LBA4404 by triparental mating (8). Plant Transformation, Regeneration, and Growth. Agrobacterium strains carrying plasmid pSpA or pGZ322 were used to infect leaf discs from Petunia hybrida line V23 x R5 1. Leaf disc infections, the selection of kanamycin-resistant calli, and the regeneration of transgenic plants were carried out as described by Horsch et al. (14) with the following modifications. Leaf discs were preincubated for 3 d on preincubation medium (14). Fully expanded leaf discs were inoculated with Agrobacterium and cocultivated for 3 d on preincubation medium prior to transfer to selective medium. Feeder-layer plates were not used. Transformed plants were transferred to potting soil and grown in a growth chamber at 20°C with a 16 h photoperiod. Flowers were self-pollinated and seed was collected at 10, 16, 19, and 22 dpp. Analysis of DNA and RNA. Total DNA was extracted from leaves oftransformed and untransformed Petunia by the method of Dellaporta et al. (7). The procedure of Southern (32) was used to detect the presence of the zein chimeric gene. Total DNA (15 ,ug) was digested with EcoRI and HindIII, separated by electrophoresis through a 1.2% agarose gel, and transferred to nitrocellulose. The blots were hybridized with a 1.7 kbp EcoRI-HindIII fragment containing the phaseolin 5' and zein coding sequences of pSpA. This same fragment was used for gene copy number reconstructions. Hybridization and autoradiography were performed as described by Goldsbrough et al. (1 1). Total RNA was extracted from leaves, stems, roots, immature petals, and seeds of Petunia essentially as described by Beachy et al. (3). After the final phenol extraction, total nucleic acids were precipitated by the addition of two volumes of ethanol, and total RNA was separated from DNA by precipitation from 2 M LiCl. Total RNA (10 ,g) was separated by electrophoresis through 1.2% formaldehyde-agarose gels and transferred to nitrocellulose (33). The 19 kD zein RNA used for size and quantitation standards was made by the in vitro transcription of plasmid
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WILLIAMSON ET AL.
pSP6.cZl9cl (9) with SP6 RNA polymerase. Hybridization conditions and autoradiography were as described for DNA analyses. Protein Extraction and Analysis. Protein was extracted from Petunia seeds by the method of Osborne (24). Seeds from 10 pods (about 250 mg of seed) were ground to a fine powder in liquid nitrogen and extracted sequentially with 0.5 M NaCl (fraction I), 60% isopropanol (fraction II), and 2.3% SDS (fraction III). The protein in each fraction was quantitated by the method of Bradford (5). Total protein per pod-equivalent of seed was estimated by adding the protein present in fractions I through III. Total alcohol-soluble protein (fraction II) from the equivalent of one pod of seeds (about 50 ,ug of protein) was separated by electrophoresis through a 12.5% SDS-polyacrylamide gel (18) and electrophoretically transferred to nitrocellulose (34). Purified a-zeins from maize endosperm were used as mol wt and quantitation standards. Blots were incubated with polyclonal rabbit anti-a-zein serum followed by goat anti-rabbit antiserum coupled to horseradish peroxidase (Bio-Rad). An immunoperoxidase kit (Bio-Rad) was used to assay horseradish peroxidase activity.
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RESULTS
Construction of Chimeric Genes Containing the 19 kD Zein Coding Sequence. In previous studies of zein gene expression in transgenic plant tissues (1 1, 23, 35), zein mRNA was present at low levels and zein protein was not detected. These results could have been due to a number of factors including inefficient utilization of the zein promoter sequences in dicot tissues, early termination of transcription, or instability of the mRNA. To distinguish among these, the chimeric genes depicted in Figure 1 were constructed. A phaseolin promoter was chosen to direct the transcription of the 19 kD zein coding sequence because high levels of transcription of the phaseolin gene have been demonstrated in dicot seeds (30). The zein coding sequence used in these constructions is derived from a cDNA clone and does not contain a polyadenylation consensus sequence. The cDNA was, therefore, linked to polyadenylation signal sequences from two sources. The clone pSpA incorporates the polyadenylation signal sequence from a phaseolin gene, whereas pGZ322 contains the polyadenylation signal sequence from a gene encoding a 15 kD zein. Plant Transformations. The chimeric gene constructs were subcloned into the binary Ti-plasmid vectors pBinl9 and pGA492, mobilized into Agrobacterium tumefaciens LBA4404 by triparental matings (8), and the resulting transconjugants used to infect Petunia leaf discs (14). Regenerated plants showing kanamycin resistance were analyzed by Southern blot analysis to confirm transformation. Nine independent transformants, containing from 2 to10 copies of the unrearranged chimeric zein genes, were chosen for further study (Fig. 2A). Analysis of Zein Expression in Transgenic Plants. The expression of the chimeric genes was analyzed by Northern and Western blot analyses of total RNA and alcohol-soluble protein, respectively (Fig. 2, B and C). For Northern blot analysis,10 jig of total RNA from 19 dpp seed from each transformed plant was separated by formaldehyde-agarose gel electrophoresis and transferred to nitrocellulose (Fig. 2B). The blots were hybridized with a 1.7 kbp EcoRI-HindIII fragment from pSpA that contains the entire zein coding sequence plus the phaseolin promoter (Fig. 1B). Plants transformed with either pSpA or pGZ322 contained RNA of the expected size that was not present in untransformed Petunia (Fig. 2B). Comparison of DNA and RNA blots (Fig. 2, A and B) showed little or no correlation between the zein gene copy number and the steady state RNA level. For example, plant pSpA-4 contains three to four copies of the zein gene but makes relatively little zein mRNA when compared to plant pSpA- 12, which contains two to three copies of the gene (Fig. 2, A and B, lanes 5 and 8). The steady state level of zein mRNA in transgenic
Plant Physiol. Vol. 88, 1988
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FIG. 2. Analysis of 19 kD zein expression in transgenic Petunia. A, blot analysis. DNA (10 from each transformed plant and from untransformed Petunia was digested with EcoRI and HindIII, separated by agarose gel electrophoresis and transferred to nitrocellulose. Blots were hybridized with the 1.7 kbp EcoRI-HindIII fragment from pSpA (Fig. 1). Lanes 1 to 3 contain 10, 5, and 1 gene-copy reconstructions, respectively; lanes 4 to 12 contain DNA from plants pSpA-1, -4, 6, -11 and -12, pGZ322-41, -59, -68, and -69, respectively; lane 13 contains DNA from untransformed Petunia. B, Northern blot analysis. Total RNA (10,g) from 19 dpp seed was separated by formaldehydeagarose gel electrophoresis, transferred to nitrocellulose and hybridized as in (A). Lanes1 to 3 contain 1, 0.5, and 0.1 ng, respectively, of zein RNA prepared by in vitro transcription of the plasmid pSP6.cZ19cl.Awt with SP6 RNA polymerase (Fig. 1). Lanes 4 to 13 are as in (A). C, Western blot analysis. Total alcohol-soluble protein (about 40Mug) from one pod-equivalent of 22 dpp seeds (total protein from one pod-equivalent of seed is about 500 Mg) was separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose. Blots were initially reacted with anti-a-zein polyclonal antibodies, followed by reaction with a second antibody coupled to horseradish peroxidase. Lanes1 to 3 contain 10, 5, and1 ng, respectively, of purified a-zein (19 and 22 kD). Lanes 4 to 13 are as in (A) and (B).
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total seed RNA, or approximately 0.1 to 1.0% of the seed RNA. Zein mRNA levels in plants transformed with pGZ322, containing the 15 kD zein 3' flanking sequence, do not greatly differ from those in plants transformed with pSpA. Western blotting was used to detect the 19 kD zein protein in the seeds of these plants. Because zeins are alcohol-soluble, total alcohol-soluble protein from one pod-equivalent of 22 dpp seed from each transformed plant was separated by SDS-PAGE and transferred to nitrocellulose. The blots were incubated with an anti-zein polyclonal antibody and the resulting complexes visualized by reaction with a second antibody coupled to horseradish peroxidase (Fig.2C). Seed from the transformed plants contained a 19 kD cross-reacting protein not present in the seed from untransformed plants. This protein comigrated on SDS polyacrylamide gels with the mature form of the zein protein, indicating that the signal peptide is apparently recognized and cleaved in Petunia seeds. The level of zein protein was higher in those plants with higher zein mRNA levels. However, variation in protein levels was, at most, 2- to 3-fold, while variation in RNA levels was up to 10-fold. In addition, the steady state levels
poly(A)+
SYNTHESIS OF ZEIN PROTEIN IN PETUNIA of 19 kD zein protein were very low compared to the amount of zein mRNA (see "Discussion"). Developmental and Tissue-Specific Expression of the Chimeric 19 kD Zein Gene. One plant, pSpA- 1, was chosen for more detailed analysis. Northern blot analysis of total RNA from leaves, stems, roots, petals, and seeds indicated that the expression of the phaseolin/zein chimeric gene was seed-specific (Fig. 3). Total RNA from the leaves of all pSpA transformed plants was also analyzed and found not to contain detectable levels of zein mRNA (data not shown). Total RNA from seed at 19 dpp from plant gZl9, a plant containing a 19 kD zein genomic with about 850 bp 5' flanking sequence, was included in this analysis. Although zein RNA was detected in the seed of this plant by S1 nuclease analysis (35), levels are too low to be detected by these Northem analyses. The temporal regulation of both zein mRNA and protein accumulation was analyzed in developing seeds. Total RNA and alcohol-soluble protein extracted from developing seeds at 10, 16, 19, and 22 dpp were analyzed by Northern and Western blotting, respectively. The results shown in Figure 4A indicate that zein mRNA began to accumulate between 10 and 16 dpp, peaked around 19 dpp, and was present at lower levels in the mature seed at 22 dpp. Zein protein also appeared at approximately 16 dpp and accumulated thereafter to reach a maximum in mature seeds (Fig. 4B). The expression of the 19 kD zein polypeptide is, therefore, temporally regulated during Petunia seed development in a manner consistent with the results previously reported for the expression of a phaseolin gene in transgenic tobacco plants (30). The accumulation of the 19 kD zein protein in seeds of these transgenic Petunia plants was much lower than one would expect from the high steady state levels of zein mRNA (Figs. 2 and 4). Zein mRNA comprised up to 1% of the total poly(A)+ RNA population in Petunia seed (about 1 ng/10 ,ug total RNA, Fig. 2B). This is comparable to levels of phaseolin and soybean ficonglycinin mRNA measured in transgenic tobacco and Petunia, respectively (3, 30). These proteins, however, made up from 1 to 3% of the total seed protein, while the 19 kD zein protein was only 0.001 to 0.005% of the total seed protein (1-5 ng/500 ,ug total protein, Fig. 2C). This observation suggests that translational or posttranslational processes such as protein stability, transport, or localization might play an important role in zein accumulation in Petunia.
DISCUSSION In previous studies of the expression of the 19 kD zein gene under the regulation of its own 5' and 3' flanking sequences, only small amounts of mRNA were found and the corresponding protein could not be detected in transgenic dicotyledonous plants or tumors (1 1, 23, 35). The low steady state levels ofzein mRNA observed in these studies could have been due either to the lack cr Iw
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of recognizable upstream transcriptional regulatory sequences or to instability of the resulting transcript. Likewise, the absence of detectable protein could have been the result of inefficient translation or a posttranslational mechanism, such as instability of the zein polypeptide itself. We constructed phaseolin-zein transcriptional fusions to facilitate the analysis of those factors affecting zein expression in Petunia. Both transcriptional fusions, under the direction of a dicot promoter, resulted in high steady state levels ofzein mRNA in the seed of transformed Petunia plants (Fig. 3A). The variation of zein mRNA levels during seed development was also consistent with results previously reported for the expression of the phaseolin gene in dicots (Fig. 4A) (30). These results suggest that sequences within the zein mRNA do not promote its instability in transgenic Petunia plants. In addition, because this mRNA is not spliced (26), the differences in steady state levels of RNA transcribed from genomic clones ([35]; Fig. 3) and cDNA fusions (this study) cannot be due to degradation of unspliced intermediates. Finally, our results suggest that the dicot and monocot polyadenylation sequences in these constructions are utilized with approximately equal efficiency in Petunia embryo tissue. The low steady state levels of zein mRNA detected in transgenic plants containing zein genomic sequences most likely result, therefore, from poor recognition of upstream activating sequences in Petunia, at least in those organs and cell types examined in this study. In this study, zein protein accumulated in the seed of all transformed plants and reached a maximum in mature seed (Fig. 4B). The signal peptides of the proteins appeared to be recognized and cleaved, and plants with higher zein mRNA levels tended to show higher zein protein levels (cf. Figs. 2B and 2C). Zein protein levels were, however, much lower than would be predicted from the steady state zein mRNA levels, suggesting that translational or post-translational processes such as protein stability, processing, transport, or localization might play an important role in zein accumulation in Petunia. Preliminary in situ localization studies (data not shown) suggest that zein protein accumulates in dispersed networks or in small aggregates at the cell surface. Although inefficient translation or specific protein turnover might contribute to the low zein protein levels, this preliminary result suggests that transport and targeting to protein bodies might also play an important role in storage protein accumulation. Protein bodies containing the major storage proteins of dicots differ in both ontogeny and localization from the zeincontaining protein bodies in maize. In maize, the zeins are stored in RER-derived protein bodies in endosperm cells (19, 20). In dicots, the storage proteins are primarily found in cotyledon cells where, after synthesis on the RER, they are transported via the golgi to vacuole-derived storage vesicles (21, 31). It is, therefore, reasonable that the successful transgenic expression of proteins, in addition to requiring specific transcriptional regulatory seFIG. 3. Analysis of transcription of the phaseolin-zein chimeric gene in tissues and organs of transgenic Petunia plants. Total RNA (10 Aig) from 19 dpp seed, lane 4; leaf, lane 5; immature petal, lane 6; root, lane 7 and stem, lane 8, of plant pSpA-l; seed from plant gZ 19, lane 9, transformed with the native zein genomic sequence pgZ19abl (Ueng et al., 1988); and RNA from control untransformed Petunia seeds, lane 10, was separated by formaldehyde-agarose gel electrophoresis and transferred to nitrocellulose. The resulting Northem blots were hybridized with the 1.7 kbp EcoRI-HindIII fragment from clone pSpA (Fig. 1). Lanes 1 to 3 contain 1, 0.5, and 0.1 ng, respectively, of zein RNA prepared by the in vitro transcription of clone pSP6.cZI9cl.Awt with SP6 RNA polymerase (Fig. 1).
1006
WILLIAMSON ET AL. C
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The absence of detectable zein precursor polypeptides in Petunia cotyledon cells suggests that the protein is probably made on and translocated into the RER, because the enzyme responsible for cleaving signal peptides is only active in the lumen of the RER (16). If these mature zein proteins contain peptide signals causing their retention in the RER in maize, it is possible that these signals are not correctly recognized in Petunia embryo
tissues. Two recent studies have utilized similar strategies to elicit zein
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chimeric gene containing a phaseolin promoter linked to a 15 kD zein coding sequence. High levels of embryo-specific expression of the 15 kD zein protein resulted. Zein, however, was localized in what appeared to be typical dicot storage bodies in the embryo rather than in the RER-derived protein bodies characteristic of maize endosperm. Because the primary, as well as proposed secondary, structures of the 15 and 19 kD zeins are quite different (31), this difference in routing, compartmentation and stabilization in dicot embryo tissue is perhaps not surprising. Schernthaner et al. (29) introduced into tobacco two chimeric genes containing 19 or 23 kD zein coding sequences transcribed from CaMV 35S promoters. The accumulation of both zein proteins was observed in the endosperm of the resulting transgenic plants. Although the 35S promoter theoretically allows transcription of these chimeric genes in both embryo and endosperm tissue, neither zein protein was detected in the tobacco embryos.
a
Plant Physiol. Vol. 88, 1988
involved in controlling transcription, splicing, and polyadenylation vary from organism to organism (15, 17). Taken together with our data, these results suggest that the sequence recognition mechanisms involved in the efficient translation, routing, and compartmentation of proteins might vary, not only between monocots and dicots, but also in different tissues within the plant. Analysis of these similarities and differences in heterologous systems offers an excellent opportunity to identify important control points in gene expression and to identify sequences involved in that control. Acknowledgments-The authors are indebted to Marlaya Wyncott, Gary Thompson, Patti Sherril, Mark Golub, Kelly Roth, Richard Wilson, and Judy Lindell for excellent technical assistance, and to Wilma Foust for preparation of this manuscript. We wish to thank Dr. Al Kriz for rabbit antisera and Dr. Les Hoffman for the clone p8.8.pro. We gratefully acknowledge Kathryn L. Shaw, Olympia Mejia, Craig Lending, Dr. Charles Bracker, and the staff of the Electron Microscopy Center in Agriculture at Purdue University for their invaluable assistance.
LITERATURE CITED 1. AN G 1986 Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. Plant Physiol 81: 86-91 2. ARGOS P, K PEDERSON, MD MARKS, BA LARKINS 1982 A structural model for maize zein proteins. J Biol Chem 257: 9984-9990 3. BEACHY RN, Z-L CHEN, RB HORSCH, SG ROGERS, NL HOFFMANN, RT FRALEY 1985 Accumulation and assembly of soybean ,B-conglycinin in seeds of transformed petunia plants. EMBO J 4: 3047-3053 4. BEVAN M 1984 Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12: 8711-8721 5. BRADFORD M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem 72: 248-254 6. DAS OP, JW MESSING 1987 Allelic variation and differential expression at the 27-kilodalton zein locus in maize. Mol Cell Biol 7: 4490-4497 7. DELLAPORTA SL, J WOOD, JB HICKS 1983 A plant DNA minipreparation: version II. Plant Mol Biol Rep 1/4: 19-21 8. DITTA G, S STANFIELD, D CORBIN, DR HELINSKI 1980 Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank in Rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347-7351 9. GALILI G, EE KAWATA, RE CUELLAR, LD SMITH, BA LARKINS 1986 Synthetic oligonucleotide tails inhibit in vitro and in vivo translation of SP6 transcripts of maize zein cDNA clones. Nucleic Acids Res 14: 1511-1524 10. GREENWOOD JS, MJ CHRISPEELS 1985 Correct targeting of the bean storage protein phaseolin in the seeds of transformed tobacco. Plant Physiol 79: 6571 11. GOLDSBROUGH PB, SB GELVIN, BA LARKINS 1986 Expression of maize zein genes in transformed sunflower cells. Mol Gen Genet 202: 374-381 12. HAGEN G, I RUBENSTEIN 1981 Complex organization of zein genes in maize.
Gene 13: 239-249 13. HOFFMAN LM, DD DONALDSON, R BOOKLAND, K RASHKA, E HERMAN 1987 Synthesis and protein body deposition of maize 15-kd zein in transgenic tobacco seeds. EMBO J 6: 3213-3221 14. HORSCH RB, JE FRY, NL HOFFMANN, D EICHHOLTZ, SG ROGERS, RT FRALEY 1985 A simple and general method for transferring genes into plants. Science
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