| The SUPERMAN-like zinc finger protein is a kind of single zinc finger protein that belong to theCys2His2-type zinc finger protein family, the conserved domains are the QALGGH amino acid residues atN-terminus and a leucine-rich motif L(I)DLXLR(K)L in the carboxy-terminal region. The superman family play animportant role in flora organ morphogenesis, embryogenesis, seed development, lateral root formation and branchingof plant.In this study, using plant Lotus japonicus as subjects, we found a new SUP-family gene, named as ZFP3involving in the regulation of root and nodule development, in Lotus japonicas databaseAnd then we used sequence comparison, homology analysis, promoter analysis and genetic transformation method to study its protein structure,, expression patterns and physiological functions, The main research results are as follows:1. Using the SUPERMAN-like protein sequences in Arabidopsis thaliana soybean, petunia and other plant organisms to blast the L. japonicasdatabase, we found a new SUP-family gene, designated as ZFP3, possibly involved in the regulation of nodule development.The SUPERMAN-like zinc finger protein belong to the TFIIIA transcription factor with a classical C2H2-type zinc finger domain and conserved QALGGH amino acid residues,closely related to plant growth and development.2. Sequence analysis in PLACE database showed that LjZFP3gene contained al.82kbnucleotides promoter region, and several important upstream promoter elements. The conserved cis-acting sequence5’-AAAGAT-3’controlling nodulin gene expression is present at its promoter region. There are four cis-acting element motifs5’-CTCTT-3’related to late nodulin genes and four regulatory elements5’-ATATT-3’responsible for root-specific expressionin the5’-upstream of the ZFP3.The analysisseems to imply that ZFP3may be a root-specific and nodule-specific gene.3. To investigate the spatial pattern of ZFP3expression,we produce transgenic hairy roots containing fusions of the promoter region of ZFP3to the β-glucuronidase (GUS) reporter gene by hair root transformation system of L. japonicus. Histochemical staining shows that GUS reporter activity is observed mainly in root vascular bundles,and no expression of ZFP3was observed in nodule primordial The intensity of blue reaction is gradually increased in the developing nodules,and is very weak in mature and senescentnodules.The Real-Time PCRassay shows ZFP3mRNA is expressed in the roots of L. japonicus before and after inoculation with M. loti, the level of2d after inoculation is much higher, as compared with that in uninoculated control roots loti and then it tends to be stable; the expression level of ZFP3was elevated in nodules from different time remained similar level. These observations confirm that the ZFP3gene is indeed expressed in roots and nodules, and indicates that ZFP3maybe involve in the process of root and nodule development in L. japonicus.4.Significant decreases in Nodulation and root growth defects were observed in ZFP3RNAi roots compared with the control hairy roots.Thus, ZFP3may act as a positive regulator in the process of root growth and nodule development in L. japonicus.5. ZFP3was expressed under the control of the maize ubiquitin promoter (ZFP3OX) in transgenic hairy roots of L. japonicus. The differences of nodule numbers per root system between the control and ZFP3OX hairy roots were not shown to be statistically significant. But the proportion of mature nodulesin ZFP3OX was higher than the control.These results suggest that ZFP3may play an important role in the nodule development.6. Recent studies have shown that C2H2-type zinc finger protein can be the target substrates of MPKS.Using yeast two-hybrid assay in vivo, we find that ZFP3are not only able to interact with several MAPKs, but also with MAPKK, implying that ZFP3may play an important role in the nodule and root development through MAPK cascades. |
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