Abstract
Tomatoes (Lycopersicon esculentum Mill cv. Ailsa Craig) were transformed with a gene construct having 244 bp of the 5′ end of a polygalacturonase (PG) cDNA, coding for a 71 amino acid N-terminal extension to the mature protein, fused to 1320 bp of a pectinesterase (PE) cDNA encoding the full sequence of the mature PE protein. This chimaeric gene was inserted in a sense orientation between a CaMV 35S promoter and terminator for constitutive expression. In transformed tomato plants expression of the endogenous PG and PE genes in the fruit was inhibited; there was little or no observable PG and PE mRNA and a substantial reduction in the level of PG and PE enzyme activity. The transgene was expressed in the leaves of the transformed plants as demonstrated by the accumulation of mRNA, but no protein product could be identified. However, no transgene mRNA or protein were observed in the transgenic fruit.
This paper represents the first report of the down-regulation of two non-homologous endogenous genes using a single gene construct. A sense gene construct was responsible for these effects. These findings are discussed in relation to possible mechanisms of action of co-suppression.
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References
Bevan MW: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).
Bird CR, Smith CJS, Ray JA, Moureau P, Bevan MW, Bird AS, Hughes S, Morris PC, Grierson D, Schuch W: The tomato polygalacturonase gene and ripening specific expression in transgenic plants. Plant Mol Biol 11: 651–662 (1988).
Chrispeels MJ: Sorting the proteins in the secretory system. Annu Rev Plant Physiol Plant Mol Biol 42: 21–53 (1991).
Clarke BC, Moran LB, Appels R: DNA analysis in wheat breeding. Genome 32: 334–339 (1989).
Dellapenna D and Bennett AB: In vitro synthesis and processing of tomato fruit polygalacturonase. Plant Physiol 86: 1057–1063 (1988).
Fillatti JJ, Kiser J, Rose R, Comai L: Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Bio technology 5: 726–730 (1987).
Grierson D, Tucker GA, Keen J, Ray J, Bird CR, Schuch W: Sequencing and identification of a cDNA clone for tomato polygalacturonase. Nucl Acids Res 14: 8595–8603 (1986).
Grierson D, Fray RG, Hamilton AJ, Smith CJS, Watson CF: Does co-suppression of sense genes in transgenic plants involve antisense RNA. Trends Biotechnol 9: 122–123 (1991).
Hall LN, Tucker GA, Smith CJS, Watson CF, Seymour GB, Bundick Y, Boniwell JM, Fletcher JD, Ray JA, Schuch W, Bird CR, Grierson D: Antisense inhibition of pectinesterase gene expression in transgenic tomatoes. Plant J 3: 121–129 (1993).
Harriman RW, Tieman DM, Handa AK: Molecular cloning of tomato pectin methylesterase gene and its expression in Rutgers, ripening inhibitor, nonripening and never-ripe tomato fruits. Plant Physiol 97: 80–87 (1991).
Jorgensen R: Altered gene expression in plants due to trans interactions between homologous genes. Trends Biotechnol 8: 340–344 (1990).
Jorgensen R: Beyond antisense: how do transgenes interact with homologous plant genes. Trends Biotechnol 9: 266–267 (1991).
Mol JNM, van derKrol AR, vanTunen AJ, vanBlokland R, deLange P, Stuitje AR: Regulation of plant gene expression by antisense RNA. FEBS Lett 268: 427–430 (1990).
Mol JNM, vanBlokland R, Kooter J: More about cosuppression. Trends Biotechnol 9: 182–183 (1991).
Napoli C, Lemieux C, Jorgensen R: Introduction of chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279–289 (1990).
Nelson N: A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153: 375–380 (1944).
Pietrzak M, Shillito RD, Hohn T, Potrykus I: Expression in plants of two antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucl Acids Res 14: 5857–5868 (1986).
Pogson BJ, Brady CJ, Orr GR: On the occurrence and structure of subunits of endopolygalacturonase isoforms in mature green and ripening tomato fruit. Aust J Plant Physiol 18: 65–79 (1991).
Pogson BJ, Seymour GB, Brady CJ, Jones M, Goodchild D: Immunolocalisation of pectinases in tomato fruit. NZ J Crop Hor Sci 20: 137–146 (1992).
Pressey R, Woods R: Purification and properties of two pectinesterases from tomatoes. Phytochemistry 31: 1139–1142 (1992).
Ray J, Knapp J, Grierson D, Bird C, Schuch W: Identification and sequence determination of a cDNA clone for tomato pectinesterase. Eur J Biochem 174: 119–124 (1988).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Seymour GB, Harding SE, Taylor AJ, Hobson GE, Tucker GA: Polyuronide solubilization during ripening of normal and mutant tomato fruit. Phytochemistry 26: 1871–1875 (1987).
Seymour GB, Lasslett Y, Tucker GA: Differential effects of pectolytic enzymes on tomato polyuronides in vivo in vitro. Phytochemistry 26: 3137–3139 (1987).
Sheehy RE, Kramer M, Hiatt WR: Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc Natl Acad Sci USA 85: 8805–8809 (1988).
Smith CJS, Slater A, Grierson D: Rapid appearance of an mRNA correlated with ethylene synthesis encoding a protein of molecular weight 75 000. Planta 168: 94–100 (1986).
Smith CJS, Watson CF, Ray J, Bird CR, Morris P, Schuch W, Grierson D: Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334: 724–726 (1988).
Smith CJS, Watson CF, Morris P, Bird CR, Seymour GB, Gray JE, Arnol C, Tucker GA, Schuch W, Harding S, Grierson D: Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol Biol 14: 369–379 (1990).
Smith CJS, Watson CF, Bird CR, Ray J, Schuch W, Grierson D: Expression of a truncated tomato polygalac-turonase gene inhibits expression of the endogenous gene in transgenic plants. Mol Gen Genet 224: 477–481 (1990).
Tucker GA, Robertson NG, Grierson D: Changes in polygalacturonase isoenzymes during the ripening of normal and mutant tomato fruit. Eur J Biochem 112: 119–124 (1980).
Tucker GA, Grierson D: Synthesis of polygalacturonase during tomato fruit ripening. Planta 155: 64–67 (1982).
Tucker GA, Robertson NG, Grierson D: Purification and changes in activity of tomato pectinesterase isoenzymes. J Sci Food Agric 33: 396–400 (1982).
Tucker Ga, Grierson D: Fruit ripening. In: Davies DD (ed) The Biochemistry of Plants, vol 12, pp. 265–318. Academic Press, London (1987).
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Seymour, G.B., Fray, R.G., Hill, P. et al. Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct. Plant Mol Biol 23, 1–9 (1993). https://doi.org/10.1007/BF00021414
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DOI: https://doi.org/10.1007/BF00021414