臺灣博碩士論文加值系統

本研究觀察到在蕃茄果實中除了polygalacturonase及pectin methylesterase兩個主要表現的酵素之外，另有一分子量15 kDa的蛋白質其表現量亦極為顯著。由於該蛋白質的生物功能至今不詳，在此將其命名為Tomato fruit 15-kDa protein，並簡稱為TF15蛋白質。
TF15蛋白質普遍存在於各不同品系的蕃茄果實內，且於各不同發育時期皆有表現。為了選殖TF15蛋白質的生合成基因，本研究製備了蕃茄果實的cDNA library，並藉專一抗體從中選殖出HL4及HL5兩個cDNA clones。其中HL5全長為740 bp，內含一個333 bp的完整讀序，並可轉譯出110個氨基酸。TF15蛋白質的氨基酸序列中以His、Glu、Lys等三個氨基酸的含量最為豐富，分別佔全部氨基酸的19.09 %、17.27 % 及16.36 %。在該蛋白質中親水性氨基酸與疏水性氨基酸以短片段長度重複鑲嵌，形成a-helix的二級結構。在基因庫內進行比對的結果，發現TF15蛋白質與前人報導的ASR蛋白質 (Iusem et al., 1993) 有顯著的相似性。
相較於植株的根、莖、葉片等器官，TF15蛋白質在蕃茄植株中主要表現於果實部位。此外，TF15蛋白質也會受到PEG滲透逆境或ABA的處理而有短暫性之輕微誘導表現。蕃茄果實在以1 % Ethephon進行人工催熟時，TF15蛋白質的表現量並不隨著Ethephon處理時間的增加而有所改變。且無論是對乙烯不敏感的Never Ripe突變種或是對乙烯敏感的亞蔬CL5915-93D4-1-0-3品種，其果實內TF15蛋白質的表現量均為一致，故推論TF15蛋白質的表現不受荷爾蒙乙烯的影響。此外，雖然TF15蛋白質之基因表現量會隨著果實的成熟而有逐漸增加的趨勢，但其蛋白質表現量卻在果實發育至MG時期後，即已臻穩定狀態，此一結果顯示在蕃茄果實中，TF15蛋白質的生合成基因之表現，可能在post-transcriptional的層次上具有調控的機制。

In this study, we observed in tomato fruit the presence of a predominant 15-kDa protein, in addition to polygalacturonase and pectin methylesterase. Since the biological function of this protein is not yet known, here we named it as tomato fruit 15-kDa protein (TF15 protein).
TF15 protein can be detected in tomato fruits from various cultivars and of different developmental stages. To identify its encoding gene, we have constructed a tomato fruit cDNA library and selected from it two positive clones, HL4 and HL5, using an antiserum directed to the TF15 protein. The HL5 clone is 740 base pairs in length and contains an open reading frame encoding a polypeptide of 110 amino acids. The translated polypeptide is rich in His (19%), Glu (17%), and Lys (16%). Beside, the hydrophilic and hydrophobic amino acids within this polypeptide are present as intervened segments, suggesting the existence of an a-helix secondary structure. The result of a protein database search indicates the TF15 protein is high homologous to the ASR protein (Iusem et al., 1993).
Comparative to its levels in the root, stem, and leaves of tomato plant, TF15 protein is mainly expressed in the fruit. In the stem, however, the expression of TF15 protein may be transiently and slightly induced by PEG or ABA treatment. While Ethephon treatment lead tomato fruits of mature-green stage to ripened, the level of TF15 protein in these fruits remained unchanged. In addition, the levels of TF15 protein in both ethylene-sensitive (AVDRC CL5915-93D4-1-0-3) and ethylene-insensitive (Never ripe) tomato fruits were indistinguishable. These results indicate the expression of TF15 protein is not ethylene-stimulated. In ripening tomato fruits, the expression patterns of TF15 protein and its RNA transcript are distinct. For the former, the protein level has reached a steady state when fruits are at mature-green stage; while for the latter, the RNA level remains increasing along the ripening process. These data suggest the expression of TF15 protein and its encoding gene may be regulated at the post-transcriptinal level.

目 錄
壹、緒論1
重組蛋白質的生產1
基因轉殖植物的應用3
蕃茄的應用5
蕃茄果實內已知基因與蛋白質之表現7
蕃茄果實中主要表現的酵素以及專一表現於蕃茄 果實內的基因7
乙烯引起的蛋白質表現9
研究目的10
貳、研究材料與方法12
一、研究材料與蕃茄植株的種植12
1. 材料12
2. 蕃茄植株的種植13
3. 蕃茄果實的發育界定與樣品之收集13
4. 蕃茄植株的水耕栽培及polyethylene glycol (PEG) 與abscisic acid (ABA) 的處理15
二、蛋白質的分析16
1. 研磨緩衝液的選擇16
2. 蛋白質的萃取與濃縮16
3. 蛋白質電泳分析17
4. 蛋白質的銀染18
5. Coomassie brilliant blue的染色19
6. 蕃茄果實內蛋白質的定量19
7. TF15蛋白質的電泳釋出 (electroelution)20
8. 等焦電泳分析21
9. 免疫轉漬 (immunoblotting) 試驗23
三、TF15蛋白質微定序分析 (microsequencing)25
四、TF15蛋白質多株抗體之製備25
1. 免疫注射25
2. 多株抗體血清之分離26
五、蕃茄果實人工催熟處理27
六、TF15蛋白質的基因選殖28
1. Total RNA之萃取28
2. mRNA的純化29
3. RNA的電泳分析：32
4. cDNA library之構築33
5. cDNA library之免疫篩選42
6. 細菌的轉型作用 (transformation)45
7. 純化噬菌體DNA46
8. DNA電泳分析48
9. 純化細菌質體DNA-鹼性溶菌法 (alkaline lysis) 之小量純化 (mini prep)49
10. 純化細菌質體DNA：鹼性溶菌法 (alkaline lysis) 之大量純化 (large prep)50
七、北方轉漬法 (Northern blotting)53
1. 探針的製作53
2. 北方轉漬分析55
參、研究結果58
蕃茄果實內蛋白質的萃取58
蕃茄果實發育期間TF15蛋白質表現情形的初探59
TF15蛋白質的純化60
TF15蛋白質的微定序分析60
TF15蛋白質之多株抗體生產與其效價測定 (titer determination)61
TF15蛋白質之基因選殖62
蕃茄果實細胞RNA的萃取與mRNA的純化62
蕃茄果實cDNA library的建構與篩選63
蕃茄果實內TF15蛋白質之基因序列及其轉譯 氨基酸序列之分析65
TF15蛋白質的表現分析66
TF15蛋白質在蕃茄懸浮培養細胞與植株內的表現 情形66
TF15蛋白質在不同蕃茄果實組織的表現情形67
TF15蛋白質在不利環境下的表現情形67
蕃茄果實發育時期TF15蛋白質及其RNA表現之 再探68
乙烯與TF15蛋白質表現關係69
肆、討論71
蕃茄果實內蛋白質表現之探討與目標蛋白質的選擇71
TF15蛋白質生合成基因的選殖74
核酸探針及多株抗體的製作74
cDNA library的建構75
TF15核酸序列及氨基酸序列之分析76
TF15蛋白質的表現分析78
TF15蛋白質生物功能之探測80
總結…82
伍、參考文獻84
陸、圖表 100
附錄一、Schenk-Hildebrandt (SH) medium133
附錄二、Modified Hoagland''s solution134

Abu-Sarra AF, Abu-Goukh AA (1992) Changes in pectinesterase, polygalacturonase and cellulase activity during mango fruit repining. J Hort Sci 67: 561-568
Amitai-Zeigerson H, Scolnik PA, Bar-Zvi D (1994) Genomic nucleotide sequence of tomato Asr2, a second member of the stress/ripening-induced Asr1 gene family. Plant Physiol 106: 1699-1700
Amitai-Zeigerson H, Scolnik PA, Bar-Zvi D (1995) Tomato Asr1 mRNA and protein are transiently expressed following salt stress, osmotic stress and treatment with abscisic acid. Plant Sci 110: 205-213
Arakawa T, Chong DKX, Langridge WHR (1998) Efficacy of a food plant-based oral cholera toxin B subunit vaccine. Nature Biotech 16: 292-297
Archer DB (1994) Enzyme production by recombinant Aspergillus. Bioprocess Technol 19: 373-393
Baker SS, Wilhelm KS, Thomashow MF (1994) The 5''-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought-and ABA-regulated gene expression. Plant Mol Biol 24: 701-713
Barnes MF, Patchett BJ (1976) Cell wall degrading enzymes and the softening of senescent strawberry fruit. J Food Sci 41: 1392-1395
Bartels D, Singh M, Salamini F (1988) Onset of desiccation tolerance during development of the barley embryo. Planta 175: 485-492
Bathgate B, Purton ME, Grierson D, Goodenough PW (1985) Plastid changes during the conversion of chloroplasts to chromoplasts in ripening tomatoes. Planta 165: 197-204
Bednarek SY, Raikhel NV (1992) Intracellular trafficking of secretory proteins. Plant Mol Biol 20: 133-150
Benfey PN, Ren L, Chua N-H (1990) Combinatorial and synergistic properties of CaMV 35S enhancer subdomains. EMBO J 9: 1685-1696
Biggs MS, Handa AK (1988) Temporal regulation of polygalacturonase gene expression in fruits of normal, mutant, and heterozygous tomato genotypes. Plant Physiol 89:117-125
Biggs MS, Harriman RW, Handa AK (1986) Changes in gene expression during tomato fruit ripening. Plant Physiol 81: 395-403
Bird CR, Smith CJS, Ray JA, Moureau P, Bevan MW, Bird AS, Hughes S, Morris PC, Grierson D, Schuch W (1988) The tomato polygalacturonase gene and ripening-specific expression in transgenic plants. Plant Mol Biol 11: 651-662
Bleecker AB, Patterson SE (1997) Last exit: senescence, abscission, and meristem arrest in Arabidopsis. Plant Cell 9: 1169-1179
Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant protein, RNA, and DNA in polyacrylamide gels. Electrophoresis 8: 93-99
Brady CJ (1987) Fruit ripening. Annu Rev Plant Physiol 38: 155-178
Caddick MX, Greenland AJ, Jepson I, Krause K-P, Qu N, Riddell KV, Salter MG, Schuch W, Sonnewald U, Tomsett AB (1998) An ethanol inducible gene switch for plants used to manipulate carbon metabolism. Nature Biotechnol 16:177-180
Carey AT, Holt K, Picard S, Wilde R, Tucker GA, Bird CR, Schuch W, Seymour GB (1995) Tomato exo-(1→4)-b-D-galactanase. Isolation, changes during ripening in normal and mutant tomato fruit, and characterization of a related clone. Plant Physiol 108: 1099-1107
Carrington CM, Pressey R (1996) b-Galactosidase II activity in relation to changes in cell wall galactosyl composition during tomato ripening. J Am Soc Hortic Sci 121: 132-136
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263: 802-805
Chan M-T, Chang H-H, Ho S-L, Tong W-F, Yu S-M (1993) Agrobacterium-mediated production of transgenic rice plants expressing a chimeric a-amylase promoter/b-glucuronidase gene. Plant Mol Biol 22: 491-506
Chang S, Puryear JD, Dias DL, Funkhouser EA, Newton RJ, Cairney J (1996) Gene expression under water deficit in loblolly pine (Pinus taeda L.): isolation and charaterization of cDNA clones. Physiol Plant 97: 139-148
Chen RD, Tabaeizadeh Z (1992) Expression and molecular cloning of drought-induced genes in the wild tomato Lycopersicon chilense. Biochem Cell Biol 70: 199-206
Chiu W-L, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6: 325-330
Cordes S, Deikman J, Margossian LJ, Fischer RL (1989) Interaction of a developmentally regulated DNA-binding factor with sites flanking two different fruit-ripening genes from tomato. Plant Cell 1: 1025-1034
Dalsgaard K, Uttenthal A, Jones TD, Xu F, Merryweather A, Hamilton WDO, Langeveld JPM, Boshuizen RS, Kamstrup S, Lomonossoff GP, Porta C, Vela C, Casal JI, Meloen RH, Rodgers PB (1997) Plant-derived vaccine protects target animals against a viral disease. Nature Biotech 15: 248-252
Day AG, Bejarano ER, Buch KW, Burrell M, Lichtenstein CP (1991) Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA virus tomato golden mosaic virus. Proc Natl Acad Sci USA 88: 6721-6725
Deikman J (1997) Molecular mechanisms of ethylene regulation of gene transcription. Physiol Plant 100:561-566
Deikman J, Kline R, Fischer RL (1992) Organization of ripening and ethylene regulatory regions in a fruit-specific promoter from tomato (Lycopersicon esculentum). Plant Physiol 100:2013-2017
DellaPenna D, Alexander DC, Bennett AB (1986) Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening. Proc Natl Acad Sci USA 83: 6420-6424
DellaPenna D, Lincoln JE, Fischer RL, Bennett AB (1989) Transcriptional analysis of polygalacturonase and other ripening associated genes in Rutgers, rin, nor and Nr tomato fruit. Plant Physiol 90: 1372-1377
Duan X, Li X, Xue Q, Abo-Ei-Saad M, Xu D, Wu R (1996) Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotech 14: 494-498
Fischer RL, Bennett AB (1991) Role of cell wall hydrolases in fruit ripening. Annu Rev Plant Physiol. Plant Mol Biol 42: 675-703
Gatz C (1996) Chemically inducible promoters in transgenic plants. Curr Opin Biotechnol 7: 168-172
Gilad A, Amitai-Zeigerson H, Bar-Zvi D (1997) ASR1, a tomato water-stress regulated gene: genomic organization, developmental regulation and DNA-binding activity. Acta Hort 447: 447-453
Giovannoni JJ, DellaPenna D, Bennett AB, Fischer RL (1989) Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening. Plant Cell 1: 53-63
Goddijn OJM, Pen J (1995) Plants as bioreactors. Trends Biotechnol 13: 379-386
Goeddel DV (1990) Methods in Enzymology. 185: Gene expression technogy, Academic Press, San Diego
Goeddel DV, Kleid DG, Bolivar F, Heyneke HL, Yansura DG, Crea R, Hirose T, Kraszewski A, Itakura K, Riggs AD (1979) Expression in Escherichia coli of chemically synthesized gene for human insulin. Proc Natl Acad Sci USA 76: 106-110
Goeschl J, Rappaport L, Pratt H (1966) Ethylene as a factor regulating the growth of pea epicotyl subjected to physical stress. Plant Physiol 41: 877-884
Gray J, Picton S, Shabbeer J, Schuch W, Grierson D (1992) Molecular biology of fruit ripening and its manipulation with antisense genes. Plant Mol Biol 19: 69-87
Grierson D, Slater A, Speirs J, Tucker GA (1985) The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening. Planta 163: 263-271
Guerrero FD, Jones JT, Mullet JE (1990) Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol Biol 15: 11-26
Hadfield KA, Bennett AB (1998) Polygalacturonase : many genes in search of a function. Plant Physiol 117: 337-343
Harriman RW, Tieman DM, Handa AK (1991) 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
Harris WM, Spurr AR (1969) Chromoplasts of tomato fruits. II The red tomato. Am J Bot 56: 380-389
Hart RA, Lester PM, Relfsnyder DH, Ogez JR, Builder SE (1994) Large scale, in situ isolation of perplasmic IGF-I from E. coli. Bio/Technol 12: 1113-1117
Herbers K, Sonnewald U (1996) Xylanases. In MRL Owen, J Pen, eds, Transgenic Plants: a production system for industrial and pharmaceutical proteins, John Wiley and Sons, Chichester, pp 191-200
Hiatt A, Cafferkey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342: 76-78
Hobson GE, Nichols R, Davies JN, Atkey PT (1984) The inhibition of tomato fruit ripening by silver. J Plant Physiol 116: 21-29
Hong B, Uknes SJ, Ho TD (1988) Cloning and characterization of a cDNA encoding a mRNA rapidly-induced by ABA in barley aleurone layers. Plant Mol Biol 11:495-506
Ishizaki-Nishizawa O, Fujii T, Azuma M, Sekiguchi K, Murata N, Ohtani T, Toguri T (1996) Low-temperature resistance of higher plants is significantly enhanced by a nonspecific cyanobacterial desaturase. Nature Biotech 14: 1003-1006
Itakura K, Hiroso T, Crea R, Riggs AD, Heyneker HL, Bolibsr F, Boyer HW (1977) Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198: 1056-1063
Iusem ND, Bartholomew DM, Hitz WD, Scolnik PA (1993) Tomato (Lycopersicon esculentum) transcript induced by water deficit and ripening. Plant Physiol 102: 1353-1354
Johnson CM, Stout PP, Broyer TC, Carlton AB (1957) Comparative chlorine requirements of different plant species. Plant and Soil 8: 337-353
Johnson PR, Ecker JR (1998) The ethylene gas signal transduction pathway: a molecular perspective. Annu Rev Genet 32: 227-254
Keinanen K, Kohr G, Seeburg PH, Laukkanen M-L, Oker-Blom C (1994) High-level expression of function glutamate receptor channels in insect cells. Bio/Technol 12: 802-806
Kuby J (1994) Hybridomas and monoclonal antibody. In J Kuby, Immunology, Ed 2, WH Freeman and Company, New York, pp 157-174
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680-685
Lanahan MB, Yen HC, Giovannoni JJ, Klee HJ (1994) The never ripe mutation blocks ethylene perception in tomato. Plant Cell 6: 521-530
LaRosa PC, Chen Z, Nelson DE, Singh NK, Hasegawa PM, Bressan RA (1992) Osmotin gene expression is posttranscriptionally regulated. Plant Physiol 100: 409-415
Lawrence SD, Cline K, Moore GA (1997) Chromoplast development in ripening tomato fruit: identification of cDNAs for chromoplast-targeted proteins and characterization of a cDNA encoding a plastid-localized low-molecular-weight heat shock protein. Plant Mol Biol 33: 483-492
Lelievre J-M, Latche A, Jones B, Bouzayen M, Pech J-C (1997) Ethylene and fruit ripening. Physiol Plant 101: 727-739
Lincoln JE, Corde S, Read E, Fischer RL (1987) Regulation of gene expression by ethylene during Lycopersicon exculentum (tomato) fruit development. Proc Natl Acad Sci USA 84: 2793-2797
Lodish H, Baltimore D, Berk A, Zipursky SL, Matsudaira P, Darnell J (1995) Membrane structure: the plasma membrane.In H Lodish, D Baltimore, A Berk, SL Zipursky, P Matsudaira, J Darnell, eds, Molecular Cell Biology, Ed 3, WH Freeman and Company, New York, pp 595-632
Lund ST, Stall RE, Klee HJ (1998) Ethylene regulates the susceptible response to pathogen infection in tomato. Plant Cell 10: 371-382
Ma JK-C, Hiatt A (1996) Expressing antibodies in plants for immunotherapy. In MRL Owen, J Pen, eds, Transgenic Plants: a production system for industrial and pharmaceutical proteins, John Wiley and Sons, Chichester, pp 229-243
Markwell MK, Hass SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206-210
Martineau B, Summerfelt KR, Adams DF, DeVerna JW (1995) Production of high solids tomatoes through molecular modification of levels of the plant growth regulator cytokinin. Bio/Technol 13: 250-254
Mason HS, Ball JM, Shi J-J, Jiang X, Estes MK, Arntzen CJ (1996) Expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci USA 93: 5335-5340
Mason HS, Lam DM-K, Arntzen CJ (1992) Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci USA 89: 11745-11749
McElroy D, Jacobsen J (1995) What''s brewing in barley biotechonolgy? Bio/Technol 13: 245-249
McElroy D, Zhang W, Cao J, Wu R (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2: 163-171
McGarvey PB, Hammond J, Dienelt MM, Hooper DC, Fu ZF, Dietzschold B, Koprowski H, Michaels FH (1995) Expression of the rabies virus glycoprotein in transgenic tomatoes. Bio/Technol 13: 1484-1487
Meller J, Dobson MJ, Roberts NA, Tuite MF, Emtage JS, White S, Lowe PA, Patel T, Kingsman AJ, Kingsman SM (1983) Efficient systhesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene 24: 1-14
Mett VL, Lochhead LP, Reynolds PHS (1993) Copper-controllable gene expression system for whole plants. Proc Natl Acad Sci USA 90: 4567-4571
Meyer DJ, Afonso CL, Galbraith DW (1988) Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures. J Cell Biol 107: 163-175
Montgomery J, Goldman S, Deikman J, Margossian L, Fischer RL (1993) Identification of an ethylene-responsive region in the promoter of a fruit ripening gene. Proc Natl Acad Sci USA 90: 5939-5943
Moustacas AM, Nari J, Diamantidis G, Noat G, Crasnier M, Borel M, Ricard J (1986) Electrostatic effects and dynamics of enzyme reactions at the surface of plant cells. 2. The role of pectin methyl esterase in the modulation of electrostatic effects in soybean cell walls. Eur J Biochem 155:191-197
Nishizawa M, Ozawa F, Hishinuma F (1987) Construction of an expression and secretion vector for the yeast Saccharomyces cerevisiae. Agric Biol Chem 51: 515-521
Odell JT, Nagy F, Chua N-H (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810-812
Oeller PW, Lu M-W, Taylor LP, Pike DA, Theologist A (1991) Reversible inhibition of tomato fruits senescence by antisense RNA. Science 254: 437-440
Padmanabhan V, Dias DMAL, Newton RJ (1997) Expression analysis of a gene family in loblolly pine (Pinus taeda L.) induced by water deficit stress. Plant Mol Biol 35: 801-807
Pear JR, Ridge N, Rasmussen R, Rose RE, Houck CM (1989) Isolation and characterization of a fruit-specific cDNA and the corresponding genomic clone from tomato. Plant Mol Biol 13: 639-651
Pen J (1996) Comparison of host syetems for the production of recombinant proteins. In MRL Owen, J Pen, eds, Transgenic Plants: a production system for industrial and pharmaceutical proteins, John Wiley and Sons, Chichester, pp 149-167
Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennet WF, Yelverton E, Seeburg PH, Heyneker HL, Eodal DR, Collen D (1983) Cloning and expression of tissue plasminogen activator in E. coli. Nature 301: 214-221
Pressey R, Reger BJ (1989) Polygalacturonase in pollen from corn and other grasses. Plant Sci 59: 57-62
Prunkard D, Cottingham I, Garner I, Bruce S, Dalrymple M, Lasser G, Bishop P, Foster D (1996) High level expression of recombinant human fibrinogen in the milk of transgenic mice. Nature Biotech 14: 867-871
Puri A, Solomos T, Kramer A (1982) Parital purification and characterisation of potato pectinesterase. Food Chem 8: 203-213
Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8: 423-488
Rossi M, Carrari F, Cabrera-Ponce JL, Vazquez-Rovere C, Herrera-Estrella L, Gudesblat G, Iusem ND (1998) Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems. Mol Gen Genet 258: 1-8
Rossi M, Iusem ND (1994) Tomato (Lycopersicon esculentum) genomic clone homologous to a gene encoding an abscisic acid-induced protein. Plant Physiol 104: 1073-1074
Rossi M, Lijavetzky D, Bernacchi D, Hopp HE, Iusem N (1996) Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato. Mol Gen Genet 252: 489-492
Schenk RV, Hildebrandt AC (1972) Medium and technique for induction and growth monocotyledonous and dicotyledonous plant cell cultrue. Can J Bot 50: 199-204
Shirzadegan M, Christie P, Seemann JR (1991) An efficient method for isolation of RNA from tissue cultured plant cells. Nucleic Acids Res 19: 6055-6056
Sijmons PC, Dekker BMM, Schrammeijer B (1990) Production of correctly processed human serum albumin in transgenic plants. Bio/Technol 79: 1549-1557
Silhavy D, Hutvagner G, Barta E, Banfalvi Z (1995) Isolation and characterization of a water-stress-inducible cDNA clone from Solanum chacoense. Plant Mol Biol 27: 587-595
Singh NK, LaRosa PC, Handa AK, Hasegawa PM, Bressan RA (1987) Hormonal regulation of protein synthesis associated with salt tolerance in plant cells. Proc Natl Acad Sci USA 84 :739-743
Sitrit Y, Downie B, Bennett AB, Bradford KJ (1996) A novel exo-polygalacturonase is associated with radicle protrusion in tomato (Lycopersicon esculentum) seeds (abstract no. 752). Plant Physiol 111: S-161
Smith CJS, Watson CF, Morris PC, Brid CR, Seymour GB, Gray JE, Arnold C, Tucker GA, Schuch W, Harding S, Grierson D (1990) Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol Biol 14: 369-379
Smith CJS, Watson CF, Ray J, Bird CR, Morris PC, Schuch W, Grierson D (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334: 724-726
Steele NM, McCann MC, Roberts K (1997) Pectin modification in cell walls of ripening tomatos occurs in distinct domains. Plant Physiol 114: 373-381
Stepien PP, Brousseau R, Wu R, Narang S, Thomas DY (1983) Synthesis of a human insulin gnen. VI. Expression of the synthetic proinsulin in yeast. Gene 24: 289-297
Thelander M, Narita JO, Gruissem W (1986) Plastid differentiation and pigment biosynthesis during tomato fruit ripening. Curr Top Plant Biochem Physiol 5: 128-141
Toki S, Takamatsu S, Nojiri C, Ooba S, Anzai H, Iwata M, Christensen AH, Quail PH, Uchimiya H (1992) Expression of a maize ubiquitin gene promoter-bar chimeric gene in transgenic rice plants. Plant Physiol 100: 1503-1507
Tucker GA, Grierson D (1982) Synthesis of polygalacturonase during tomato fruit ripening. Planta 155: 64-67
Tucker GA, Robertson NG, Grierson D (1980) Changes in polygalacturonase isoenzymes during the ripening of normal and mutant tomato fruit. Eur J Biochem 112: 119-124
Tucker GA, Robertson NG, Grierson D (1982) Purification and changes in activity of tomato pectinesterase isoenzymes. J Sci Food Agric 33: 394-400
Vaidyanathan R, Kuruvilla S, Thomas G (1999) Characterization and expression pattern of an abscisic acid and osmotic stress responsive gene from rice. Plant Sci 140: 21-30
van Engelen FA, Schouten A, Molthoff JW, Roosien J, Salinas J, Dirkse WG, Schots A, Bakker J, Gommers FJ, Jongsma MA, Bosch D, Stiekema WJ (1994) Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco. Plant Mol Biol 26: 1701-1710
van Haaren MJJ, Houck CM (1991) Strong negative and positive regulatory elements contribute to the high-level fruit-specific expression of the tomato 2A11 gene. Plant Mol Biol 17: 615-630
Visser RGF, Jacobsen E (1993) Towards modifying plants for altered starch content and composition. Tre Biotech 11: 63-68
Walden R, Schell J (1990) Techniques in plant molecular biology--progress and problems. Eur J Biochem 192: 563-576
Williams DC, Frank RM, Muth WL, Burnett JP (1982) Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins. Science 215: 687-689
Willmitzer L, Toepeer R (1992) Manipulation of oil, starch, and protein composition. Curr Opin Biotechnol 3: 176-180
Wiseman A (1993) Genetically-engineered mammalian cytochromes P-450 from yeast-potential applications. Tred Biotech 11: 131-136
Xu R, Goldman S, Coupe S, Deikman J (1996) Ethylene control of E4 transcription during tomato fruit ripening involves two cooperative cis elements. Plant Mol Biol 31: 1117-1127
Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Annu Rev Plant Physiol 35: 155-189
Yang YO, Shah J, Klessig DF (1997) Signal perception and transduction in defense responses. Genes Dev 11: 1621-1639
Zhu Z, Hughes KW, Huang L (1994) Expression of human alpha-interferon cDNA in transgenic rice. Plant Cell Tiss Org Cult 36: 197-204