大豆与重氮根瘤菌的共生关系使USDA110始终与田间其他根瘤菌竞争,具有重要的农艺和环境意义。在这里,双荧光素酶报告基因测定被用来监测两种主要的缓生根瘤菌感染大豆根的动力学。更明确地说,设计了荧光素酶标记的重氮芽孢杆菌USDA110(USDA110-FLuc)和缓生根瘤菌USDA94(USDA94-RLuc),共同接种到大豆种子中,并通过生物发光成像观察它们在根瘤中的定殖。结果表明,USDA110-FLuc比USDA94-RLuc更早开始感染,但是随着植物的生长,它在结节中的占有率下降了。结瘤试验表明,nodD1突变株USDA110,包括CRISPR工程突变体,竞争力不如野生型。我构建了siRNA来敲除不同靶位点的nodD1,并将它们转化到细菌中。令人惊讶的是,尽管具有3个末端靶位点的siRNA能够抑制高达65%的nodD1表达,用生物分析仪对总RNA的分析显示,siRNA转化和野生型USDA110菌株的23S/16S-rRNA比率相似,但低于nodD1突变体。总之,目前的工作-报道了B.diazoeffiensUSDA110在大豆结节早期占据中的竞争力和基因nodD1作为这种感染的关键决定因素-提供了对微生物中siRNA沉默的见解,并展示了一种高效的成像方法,可能为许多生物学研究领域带来许多新的途径。
The symbiosis of soybean with Bradyrhizobium diazoefficiens USDA110, which always competes with other rhizobia in the field, is of great agronomic and environmental importance. Herein, a dual-luciferase reporter assay was utilized to monitor the dynamics of two dominant bradyrhizobia infecting roots of soybean. More explicitly, luciferase-tagged B. diazoefficiens USDA110 (USDA110-FLuc) and Bradyrhizobium elkanii USDA 94 (USDA94-RLuc) were designed, co-inoculated into soybean seeds, and observed for their colonization in root nodules by bioluminescence imaging. The results showed that USDA110-FLuc initiated infection earlier than USDA94-RLuc, but its occupancy in the nodules decreased as the plant grew. A nodulation test showed that nodD1 mutant USDA110 strains, including CRISPR engineered mutants, were less competitive than wild type. I constructed siRNAs to knockdown nodD1 at different target sites and transformed them into the bacteria. Surprisingly, although siRNAs - with 3\' end target sites - were able to repress up to 65% of nodD1 expression, the profiling of total RNAs with a bioanalyzer revealed that 23S/16S-rRNA ratios of siRNA-transformed and wild type USDA110 strains were similar, but lower than that of nodD1 mutant. In short, the current work - while reporting the competitiveness of B. diazoefficiens USDA110 in early occupancy of soybean nodules and the gene nodD1 as a key determinant of this infection - gives an insight on siRNA silencing in microbes, and demonstrates a highly efficient imaging approach that could entail many new avenues for many biological research fields.