Controlling the Expression of Rhizobial Genes During Nodule Development with Elements and An Inducer of the lac Operon
Controlling the Expression of Rhizobial Genes During Nodule Development with Elements and An Inducer of the lac Operon
复制标题
DOI：
10.1094/mpmi-07-10-0155
复制
发表时间：
2011-04-01
影响因子：
3.5
通讯作者：
Noel, K. Dale
中科院分区：
文献类型：
--
作者：
Box, Jodie;Noel, K. Dale
一个简单的策略进行了测试，在根瘤发育过程中施加人工调控根瘤菌基因。将异丙基-β-D-1-硫代半乳糖苷（IPTG）加入到液体根培养基中，以维持由大肠杆菌lac启动子/操纵子和阻遏基因lad控制的根瘤菌基因的表达。相反，设计冲洗方案以充分去除IPTG，使得基因在被诱导后可以被抑制。在此对照下，gusA在确定的菜豆根瘤菌和不确定的苜蓿中华根瘤菌共生体中都表现出清晰的表达和抑制。显然，IPTG以足够未降解的浓度被吸收，即使在结节的内部，基因表达也被去抑制。此外，冲洗方案导致gusA的明显抑制。重要的是，没有观察到IPTG对根瘤发育、感染或固氮的有害影响。火箭筒通过双质粒构建体将脂多糖O抗原和感染菜豆所需的etli CE 3基因置于此控制之下。当将该构建体加入到该基因中具有无效突变的菌株中时，感染、根瘤发育和固氮酶活性都取决于接种后从根部冲洗IPTG之前的时间长度。
A simple strategy was tested for imposing artificial regulation of rhizobial genes during nodule development. Isopropyl-beta-D-1-thiogalactoside (IPTG) was added to liquid root media to sustain expression of rhizobial genes controlled by Escherichia coli lac promoter/operators and repressor gene lad. Conversely, a rinsing protocol was devised to remove IPTG sufficiently that genes could be repressed after having been induced. gusA under this control exhibited clearly delineated expression and repression in both the determinate Rhizobium etli Phaseolus vulgaris and the indeterminate Sinorhizobium meliloti Medicago sativa symbioses. Apparently, IPTG was taken up in sufficiently undegraded concentrations that gene expression was derepressed even in interior portions of the nodule. Moreover, the rinsing protocol led to obvious repression of gusA. Importantly, no deleterious effects of IPTG on nodule development, infection, or nitrogen fixation were observed. An R. etli CE3 gene required for lipopolysaccharide O antigen and infection on bean was put under this control by means of a two-plasmid construct. When this construct was added to a strain with a null mutation in this gene, infection, nodule development, and nitrogenase activity all depended on the length of time before IPTG was rinsed from the roots after inoculation.