Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan.
Curr Microbiol. 2010 Nov;61(5):361-9. doi: 10.1007/s00284-010-9620-7. Epub 2010 Mar 26.
Five cyanobacterial strains, Anabaena sp. Ck1, Oscillatoria sp. Ck2, Phormidium sp. Ck3, Chroococcidiopsis sp. Ck4, and Synechosystis sp. Ck5 were selected for their positive cytokinins-like activity using cucumber cotyledon bioassay and GUS assay in Arabidopsis ARR5::GUS. Classical cucumber cotyledon bioassay was modified for direct screening of cyanobacteria avoiding need for extraction and purification. Cytokinins from cyanobacteria were absorbed onto filter paper which was then assayed for cytokinins-like activity. A rapid chromatographic method was developed for the simultaneous determination of cytokinins and indole-3-acetic acid (IAA). Cyanobacterial biomass (50-100 mg) and cell-free culture filtrate were extracted in Bieleski buffer and purified by solid-phase extraction. The extract was used to determine phytohormones by ultra performance liquid chromatography and electrospray ionization-tandem mass spectrometry in positive and negative modes, respectively, with multiple reactions monitoring. Stable isotope-labeled cytokinins and IAA standards were added in the samples to follow recovery of the compounds and method validation. Five cytokinins determined in the selected strains were Zeatin (cis and trans isomers), Zeatin riboside, Dihydrozeatin riboside, and zeatin-o-glucoside. The strains were shown to accumulate as well as release the phytohormones.
选用 5 株蓝藻，即鱼腥藻 Ck1、颤藻 Ck2、束丝藻 Ck3、微囊藻 Ck4 和聚球藻 Ck5，通过黄瓜子叶生物测定和拟南芥 ARR5::GUS 中的 GUS 测定，检测其具有阳性细胞分裂素样活性。经典的黄瓜子叶生物测定法经过改良，可直接筛选蓝藻，避免了提取和纯化的需要。蓝藻细胞分裂素被吸收到滤纸上，然后测定其细胞分裂素样活性。建立了一种同时测定细胞分裂素和吲哚-3-乙酸（IAA）的快速色谱方法。用 Bieleski 缓冲液从蓝藻生物量（50-100mg）和无细胞培养液中提取，并用固相萃取进行纯化。提取物用于通过正、负离子模式的超高效液相色谱和电喷雾串联质谱，分别采用多反应监测进行植物激素的测定。在样品中添加稳定同位素标记的细胞分裂素和 IAA 标准品，以跟踪化合物的回收率和方法验证。在选定的菌株中测定了 5 种细胞分裂素，分别为玉米素（顺式和反式异构体）、玉米素核苷、二氢玉米素核苷和玉米素-O-葡萄糖苷。这些菌株既能积累又能释放植物激素。
