Torrigiani Patrizia, Scaramagli Sonia, Ziosi Vanina, Mayer Melinda, Biondi Stefania
Dipartimento di Biologia e.s., University of Bologna, Via Irnerio 42, 40126 Bologna, Italy.
J Plant Physiol. 2005 May;162(5):559-71. doi: 10.1016/j.jplph.2004.10.008.
S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermidine and spermine synthesis through specific synthases which use putrescine, spermidine and decarboxylated S-adenosylmethionine as substrates. In order to better understand the regulation of polyamine (PA), namely spermidine and spermine, biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco (Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S promoter, by means of Agrobacterium tumefaciens and leaf disc transformation. The effect of the genetic manipulation on PA metabolism, ethylene production and plant morphology was analysed in primary transformants (R0), and in the transgenic progeny (second generation, R1) of self-fertilised primary transformants, relative to empty vector-transformed (pBin19) and wild-type (WT) controls. All were maintained in vitro by micropropagation. Primary transformants, which were confirmed by Southern and northern analyses, efficiently transcribed the antisense SAMDC gene, but SAMDC activity and PA titres did not change. By contrast, in most transgenic R1 shoots, SAMDC activity was remarkably lower than in controls, and the putrescine-to-spermidine ratio was altered, mainly due to increased putrescine, even though putrescine oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls. Despite the reduction in SAMDC activity, the production of ethylene, which shares with PAs the common precursor SAM, was not influenced by the foreign gene. Some plants were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown second generation transgenic plants, at the vegetative stage, SAMDC activity was scarcely reduced, and PA titres did not change. Finally, the rhizogenic potential of in vitro-cultured leaf explants excised from antisense plants was significantly diminished as compared with WT ones, and the response to methyl jasmonate, a stress-mimicking compound, in terms of PA conjugation, was higher and differentially affected in transgenic leaf discs relative to WT ones. The effects of SAMDC manipulation are discussed in relation to plant generation, culture conditions and response to stress.
S-腺苷甲硫氨酸脱羧酶活性（SAMDC；EC 4.1.1.21）通过特定的合酶导致亚精胺和精胺的合成，这些合酶以腐胺、亚精胺和脱羧的S-腺苷甲硫氨酸为底物。为了更好地理解多胺（PA），即亚精胺和精胺生物合成的调控，曼陀罗的SAMDC cDNA以反义方向在CaMV 35S启动子的控制下，通过根癌农杆菌和叶盘转化法导入烟草（烟草品种Xanthi）。相对于空载体转化（pBin19）和野生型（WT）对照，分析了基因操作对PA代谢、乙烯产生和植株形态在初级转化体（R0）以及自花授粉初级转化体的转基因后代（第二代，R1）中的影响。所有植株均通过微繁殖在体外保存。经Southern和Northern分析证实的初级转化体有效地转录了反义SAMDC基因，但SAMDC活性和PA含量并未改变。相比之下，在大多数转基因R1芽中，SAMDC活性明显低于对照，腐胺与亚精胺的比例发生改变，主要是由于腐胺增加，尽管腐胺氧化活性（二胺氧化酶，EC 1.4.3.6）相对于对照没有变化。尽管SAMDC活性降低，但与PA共享共同前体SAM的乙烯产生并未受到外源基因的影响。一些植株被转移到花盆中并在生长室中驯化。在这些体内生长的第二代转基因植株的营养生长阶段，SAMDC活性几乎没有降低，PA含量也没有变化。最后，与野生型相比，从反义植株上切下的体外培养叶外植体的生根潜力显著降低，并且在PA缀合方面，转基因叶盘对茉莉酸甲酯（一种模拟胁迫的化合物）的反应相对于野生型叶盘更高且受到不同影响。讨论了SAMDC操作在植物世代、培养条件和对胁迫反应方面的影响。
