Kernan A, Thornburg R W
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011.
Plant Physiol. 1989 Sep;91(1):73-8. doi: 10.1104/pp.91.1.73.
Proteinase inhibitor genes are expressed in solanaceous and leguminous plants following wounding of the foliage by mechanical methods. Previous studies have shown that a cloned proteinase inhibitor II-chloramphenicol acetyl transferase (pin2-CAT) chimeric gene is regulated in a wound-inducible manner in transgenic plants. In this study, we analyzed transgenic plant tissues for expression of the pin2-CAT gene in response to various plant hormones. We found that CAT activity was induced in tobacco (Nicotiana tabacum) callus incubated in the absence of any plant growth regulators. Addition of growth regulators to the medium thus permitted us to measure the effects of these substances on the activity of the pin2-CAT gene construction. Cytokinin (BAP) and ethylene (ethophon) even at low concentrations stimulated the expression of CAT activity by 25 to 50%. Abscisic acid at concentrations up to 4.4 x 10(-5) molar had no effect upon CAT activity, but increasing auxin (naphthalene acetic acid) levels completely inhibited the synthesis of CAT protein. Gibberellic acid had little effect except at very high concentration (2.9 x 10(5) molar). The kinetics of activation of the pin2-CAT gene were quite long (5 to 7 days) when unwounded calli were plated on media lacking auxin. This effect was documented for calli derived from several transformed plants, containing the full, chimeric pin2-CAT (pRT45) gene. In addition, calli from tissues transformed with wild-type vectors or from several plants transformed with pRT50 (a noninducible derivative of pRT45) were not induced by plating on media lacking auxin. Other naturally occurring and synthetic auxins had similar effects to naphthalene acetic acid in inhibiting the induction of the chimeric gene fusion. Finally, leaf discs from transformed plants were induced by incubation in MS liquid medium in the presence and absence of naphthalene acetic acid. NAA was also effective in down regulating the chimeric gene in whole plant tissues.
蛋白酶抑制剂基因在通过机械方法损伤叶片后，在茄科和豆科植物中表达。先前的研究表明，克隆的蛋白酶抑制剂II-氯霉素乙酰转移酶（pin2-CAT）嵌合基因在转基因植物中以伤口诱导的方式受到调控。在本研究中，我们分析了转基因植物组织中pin2-CAT基因对各种植物激素的响应表达情况。我们发现，在没有任何植物生长调节剂的情况下培养的烟草（Nicotiana tabacum）愈伤组织中，CAT活性被诱导。因此，向培养基中添加生长调节剂使我们能够测量这些物质对pin2-CAT基因构建体活性的影响。细胞分裂素（BAP）和乙烯（乙烯利）即使在低浓度下也能使CAT活性的表达提高25%至50%。浓度高达4.4×10⁻⁵摩尔的脱落酸对CAT活性没有影响，但生长素（萘乙酸）水平的增加完全抑制了CAT蛋白的合成。赤霉素除了在非常高的浓度（2.9×10⁵摩尔）下几乎没有影响。当未受伤的愈伤组织接种在缺乏生长素的培养基上时，pin2-CAT基因的激活动力学相当长（5至7天）。这种效应在源自几种含有完整嵌合pin2-CAT（pRT45）基因的转化植物的愈伤组织中得到了证实。此外，用野生型载体转化的组织或用pRT50（pRT45的非诱导衍生物）转化的几种植物的愈伤组织，在接种到缺乏生长素的培养基上时不会被诱导。其他天然存在的和合成的生长素在抑制嵌合基因融合的诱导方面与萘乙酸有相似的作用。最后，将转化植物的叶片圆盘在有无萘乙酸的MS液体培养基中培养进行诱导。萘乙酸在下调全植物组织中的嵌合基因方面也很有效。
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