Function Resrearch Of Three New Genes Isolated From Robinia Pseudoacacia In Symbiotic Nitrogen Fixation

In this research, we use the symbiotic association between Robinia pseudoacacia L. and Mesorhizobium amorphae CCNWGS0123(abbreviated as 186) as the model and three genes screened in reciprocal library using suppression subtractive hybridization as research subjects.Using q RT-PCR to analysis the dynamic expression characteristics of these three genes in root and nodule after inoculation. We obtained full-length c DNA sequence of fan84 and f107 using rapid amplification of c DNA ends(RACE) technology and constructed fan37、fan84、f107 RNA interference vector, fan84、f107 overexpression vector and fan84、f107subcellular localization vector.Through the Agrobacterium rhizogenes-mediated plant transformation experiments, we transformed recombinanted vector into plants roots, and then after inoculation of Mesorhizobium amorphae, we observed RNAi infection events under fluorescence microscopy and counted relevant data at 10 dpi.Using RT-PCR and q RT-PCR to detect silence efficiency and overexpression levels of target genes at 15 and 30 dpi.Observation on phenotypes of RNAi transgenic plants at 30 dpi and overexpressed transgenic plants at 45 dpi including plant height, root length, fresh weight and the number of nodules.Nodule paraffin section experiment was conducted using RNAi transgenic nodules at30 dpi and overexpressed transgenic nodules at 45 dpi.Through every step of the above experiments, we try to identify the functional role of these three genes in locust and symbiotic interaction process from the phenotypic and molecular level.1 The acquisition of fan84 and f107 gene full length cDNA Using rapid amplification of c DNA ends(RACE) technology, total RNA in the 15 days and 20 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 fan84 and f107 5’ and 3’ sequence, through overlapping area sequence alignment to obtain full-length c DNA sequence.2 Construction of fan84 and f107 gene subcellular localization vector and analysis of localization of protein Using ORF region of the obtained full-length c DNA to design appopriate primers and construct subcellular localization vectors.These vectors were delivered to onion epidermal cells via particle bombardment or through the Agrobacterium rhizogenes-mediated plant transformation into plants roots.We analysized the distribution of the fusion protein in onion epidermal cells and R. hairy root cell.The results showed that Fan84::GFP fusion protein widely distributed in cell cytoplasm membrane and nucleus,while F107::GFP fusion protein mainly distributed in cell membrane and nucleus.3 Construction of the fan37、fan84、f107 gene RNA interference vector and fan84、f107 gene overexpression vector and identification of the gene function We constructed fan37 、 fan84 、 f107 RNA interference vector and fan84 、 f107 overexpression vector.Through the Agrobacterium rhizogenes-mediated plant transformation experiments, we transformed recombinanted vector into plants roots and observed the impact on plant development, rhizobium infection process and nodule formation.Using RT-PCR and q RT-PCR to detect silence efficiency and overexpressed levels. We set the transformed the empty vector as control in this part of experiment. The results showed, the expression level of fan37 、 fan84 、 f107 in transformed RNAi plants roots were effectively silenced after inoculation and was overexpressed in fan84、f107 overexpression plants both at 15 and 30 dpi.fan37 and fan84 RNA interference transgenic plants showed small, root hairs short, and the number of nodules decreased significantly.f107 RNA interference transgenic plants showed small,but no significant decrease of root length and nodule number.The RNAi nodule paraffin section results showed, 30 dpi, the number and intensity of the infected cells in fan37 and fan84 RNAi nodules was far less compared to control nodule,while the infection area in f107 RNAi nodules was significantly increased which may inhibit the release of rhizobia. fan84 overexpression results showed increased root length, fresh weight but no significant changes on plant height and nodule number.f107 overexpression results showed decreased fresh weight and nodule number. The overexpressed nodule paraffin section results showed the number of infected cells was still abundant at 45 dpi compared with empty vector plant and infected cells morphology showed no signs of aging.