Ramos Humberto J O, Roncato-Maccari Lauren D B, Souza Emanuel M, Soares-Ramos Juliana R L, Hungria Mariangela, Pedrosa Fábio O
Department of Pharmacology, Biochemistry and Molecular Biology, UFPR, C. Postal 19046, CEP 81531-990 Curitiba, PR, Brazil.
J Biotechnol. 2002 Aug 28;97(3):243-52. doi: 10.1016/s0168-1656(02)00108-6.
To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.
为监测巴西固氮螺菌在小麦根际的定殖情况，我们构建了几种基于pBBR1复制子的质粒，这些质粒组成型表达绿色荧光蛋白（gfp）基因和β-葡萄糖苷酸酶（gusA）基因。这两个基因都置于庆大霉素抗性基因启动子的控制之下，从而在大肠杆菌和巴西固氮螺菌中实现高水平表达。构建的质粒即使在没有选择压力的情况下也能在巴西固氮螺菌菌株中稳定维持。我们监测了含有这些质粒的巴西固氮螺菌菌株FP2（Sp7衍生物）转接合子在无菌蛭石中受控条件下生长的小麦植株上的定殖情况。通过荧光显微镜在新鲜植物材料中很容易观察到表达绿色荧光蛋白的细菌。在幼根区域的表面，如根毛和侧根上，可以看到细胞聚集体和单个细菌。接种30天后，在侧根出现点或根表皮细胞的细胞间隙中观察到大量细胞团块。尽管我们未能在小麦根的内部皮层和木质部组织中检测到细菌，但巴西固氮螺菌内生定殖的初始阶段可能涉及本研究中检测到的位点。
