Functional Identification Of Translational Controled Tumor Protein During Symbiotic Nodulation In Robinia Pseudoacacia

Translationally controlled tumor protein(TCTP), a widespread protein in eukaryote, played an important biological role in many different cellular processes. Through analysis of different cell signaling pathway TCTP involved in, some of its biological function had been speculated or cleared. However, the function of TCTP in symbiotic nodulation on legume plants has been unknown. Rpf41, a TCTP homologous gene, identified by our lab during research of Locust symbiotic interaction process and appeared dynamic changes in the locust nodule development process. Temporal and spatial expression analysis showed that the Rpf41 genes transcription level reached maximum value at the 15 day after inoculation rhizobia in the roots of R. pseudoacacia, but the expression decreased significantly in the mature nodules. In this paper, we obtained full length cDNA sequence by RACE, and conducted the Rpf41 gene subcellular localization. We identified the function of Rpf41 gene during Locust nodule formation through RNA interference(RNAi) and overexpression. The research contents and the results are as follows: 1 The acquisition of Rpf41 gene full length cDNAUsing rapid amplification of cDNA ends(RACE) technology, total RNA in the 15 days after inoculated rhizobia in R. pseudoacacia as template to construct the 5 ’and 3’ ends of the c DNA library. According to the preliminary expriment had received gene fragments information to design gene specific primers GSP1, GSP2, amplification and get R. pseudoacacia Rpf41 5’ and 3’ sequence, through overlapping area sequence alignment to obtain full-length cDNA sequence. We conducted the conserved domain of preliminary analysis and compared sequence similarity and identity of the Rpf41 with other plant species TCTP, analysis of its evolutionary status with other species and predict its function. 2 Construction Rpf41 gene subcellular localization vector and analysis of localization of proteinWe constructed the Rpf41 gene subcellular localization vector. Using the method of onion epidermal cells instantaneous expression, we analysised the distribution of the Rpf41 protein in onion epidermal cells. The results showed that Rpf41 protein widely distributed in cell membrane, nucleus, cytoplasm and cell skeleton. The results provided a basis for further study the function of the gene function. 3 Construction of the Rpf41 gene RNA interference vector and overexpression vector and identification of the gene functionWe constructed Rpf41 RNA interference vector and overexpression vector. Through the Agrobacterium rhizogenes receptor-mediated plant transformation experiments, we transformed recombinanted vector into plants roots and observed the formation of root hairs, rhizobium infection process and its impact on plant development and nodule formation. We set the transformed the empty vector and without any vector as control, using RT-PCR and qRT-PCR to detect silence efficiency of the target gene. The results showed, the expression level of Rpf41 in transformed RNAi plants roots were effectively silenced after inoculation and was overexpresed in Rpf41 overexpression plant. Rpf41 gene RNA interference, we found plants showed small, root hairs short, thick and sparse, the number of nodules decreased significantly and most of the nodules were white invalid nodule. The RNAi nodule paraffin results showed, 28 days post-inoculation(dpi), the number and intensity of the mature infected cells in RNAi nodules was far less compared to control nodule. And the development of RNAi nodule stay in Zone II, III zone(mature nitrogen fixation zone) is not detected.Transmission electron microscopy(TEM) results show that, RNAi nodule cells only had a small amount of bacteroids, and the bacteroid significantly shrunkage and peribacteroid space enlargement in the symbiosomes of the RNAi nodule. In some infected cells, bacteroids showed sever shrinkage and deformation, even some of them showed the signs of premature aging. Rpf41 gene overexpression results showed that there were no significantly changes on plant morphology, the phenotype and number of nodule among Rpf41 overexpression experimental group, empty vector and without any vector group plants.