Functional Research Of ZFP3during Nodule Development In Lotus Japonicus

Zinc fingers perform a broad range of functions in plant’s cellular processes as transcriptional factors. LjZFP3contains one zinc finger structure, shares a highly conserved C2H2DNA binding domain near the N terminus and a leucine-rich motif at the C terminus. In this study, We researched the expression patterns, function analysis and mutant screening of ZFP3. The main results are showed below:1. We harvested the roots, nodules, stem, leaves in different time after inoculation with M.loti MAFF303099, The total RNA was isolated, then we measured the expression level of LjZFP3using real-time PCR. The results showed that LjZFP3can be detected in all the tissues. There is no significant changes at the fisrt time, but the expression level is growing higher6days later. In ordel to find out the increasement of LjZFP3is related to the nodules’development, we measured the expression level of LjZFP3in roots and nodules, it turns out that ZFP3is richer in nodules than other tissues. The construct ZFP3pro::GUS was used to detect the tissue specific of ZFP3, GUS reporter activity was observed in root vascular bundles, nodule primordials and some immature nodules, and have no activity in mature and senescent nodules.2. Subcellular localization of LjZFP3protein:LjZFP3was expressed as a fusion protein with GFP (GFP::ZFP3) and GFP alone served as control. The plasmids were delivered to Nicotiana benthamiana, and fluorescence images were taken using a confocal laser scanning microscope, GFP::ZFP3localized to the nuclear. The GFP:ZFP3was expressed in Lotus hairy roots, and the fluorescence was observed in the nuclear. We concluded that LjZFP3is a nuclear protein.3. Overexpression of LjZFP3increased the nodule number significantly, which may indicate that LjZFP3play a key role in symbiotic process. A RNAi construct was used to identify the biological function of ZFP3in the symbiotic process through hairy root transformation and the nodule number were analyzed3weeks post inoculation. The nodule number of LjZFP3in RNAi hairy roots was lower than the control hairy roots, and the infection threat formation appeared to be affected by RNAi-LjZFP3.4. We isolate two independent Ljzfp3mutants, the insertion sites of the two Ljzfp3are both in the reading frame of LjZFP3. homozygous Ljzjp3mutants of both lines usually developed nodules, but the plants growth rate is slower than the wild type. The nodule number of Ljzfp3-1have no difference with the wild type at10DAI, but it’s significantly less than the wild type at21DAI. The Ljzfp3-2mutant allele appeared to be obvious than that of Ljzfp3-1, as Ljzfp3-2mutant is significantly different with the wild type all the time.