Department of Plant Systems Biology, VIB, Technologiepark 927, Gent, Belgium.
New Phytol. 2009 Dec;184(4):851-64. doi: 10.1111/j.1469-8137.2009.03011.x. Epub 2009 Sep 1.
*In transgenic calli and different tissues of Arabidopsis thaliana plants, the in trans silencing capacity of a 35S-beta-glucuronidase (GUS) hairpin RNA construct was investigated on a target GUS gene, under the control of the 35S, a WRKY or several cell cycle-specific promoters. *GUS histochemical staining patterns were analyzed in all tissues of the parental lines and supertransformants harboring the hairpin construct. Quantitative GUS activity measurements determined GUS suppression by a 35S-GUS hairpin or inverted repeated GUS transgenes in leaves and calli. *In some supertransformants, GUS-based staining disappeared in all tissues, including calli. In most supertransformants, however, a significant reduction was found in mature roots and leaves, but residual GUS activity was observed in the root tips, young leaves and calli. In leaves of most hairpin RNA supertransformants, the GUS activity was reduced by c. 1000-fold or more, but, in derived calli, generally by less than 200-fold. The silencing efficiency of inverted repeated sense transgenes was similar to that of a hairpin RNA construct in leaves, but weaker in calli. *These results imply that the tissue type, nature of the silencing inducer locus and the differential expression of the targeted gene codetermine the silencing efficiency.
在拟南芥的转基因愈伤组织和不同组织中,研究了在 35S、WRKY 或几个细胞周期特异性启动子的控制下,35S-β-葡萄糖醛酸酶(GUS)发夹 RNA 构建体对靶 GUS 基因的反式沉默能力。在亲本系和携带发夹构建体的超转化体的所有组织中分析了 GUS 组织化学染色模式。定量 GUS 活性测量确定了 35S-GUS 发夹或反向重复 GUS 转基因在叶片和愈伤组织中对 GUS 的抑制作用。在一些超转化体中,所有组织中的 GUS 基于染色消失,包括愈伤组织。然而,在大多数超转化体中,在成熟的根和叶中发现了显著的降低,但在根尖、幼叶和愈伤组织中观察到残留的 GUS 活性。在大多数发夹 RNA 超转化体的叶片中,GUS 活性降低了约 1000 倍或更多,但在衍生的愈伤组织中,通常降低不到 200 倍。反向重复有义转基因的沉默效率与叶片中发夹 RNA 构建体的沉默效率相似,但在愈伤组织中较弱。这些结果表明,组织类型、沉默诱导基因座的性质和靶基因的差异表达决定了沉默效率。