Function Analysis Of The Genes Involved In Robinia Pseudoacacia Symbiotic Nodulation

Legume-Rhizobium symbiosis nodulation is a complex molecular dialogue process thatinvolves a series of plant genes expression regulation.Currently, with the development ofmolecular biology and genomics, as in the model legume Medicago truncatula, Lotusjaponicus and other herbs, many of the symbiotic nodulation genes have been isolated andidentified, but especially woody legumes are species such as Robinia pseudoacacia, itsinteraction with rhizobia symbiosis genes related research is rarely reported.Seven geneshave been studied in this paper,2zheng45,2zheng139,2zheng168,2zheng240,2zheng190,2zheng287and2zheng315were isolated by our laboratory through suppression subtractivehybridization from Robinia pseudoacacia root nodulation preliminary. Previous analysisshowed that these genes may have an important regulatory role in symbiotic nodulationprocess of Robinia pseudoacacia.To further confirm the function of those genes in symbioticnitrogen fixation process in Robinia pseudoacacia, gene silencing technique andoverexpression analysis were used in this paper to confirm the effects of selected genes inRobinia pseudoacacia nodulation process, and the use of gene sub-cellular localization is todetermine its location in the cell. qRT-PCR method was used to study the dynamiccharacteristics of the target gene expression in the Robinia pseudoacacia symbiotic nodulationprocess.The results will lay an importent foundation for elucidating the specific functions ofthese genes in legume-rhizobium symbiosis.The main contents and results of the experiment were as follows:The construction of five overexpression vectors of the P-45, P-139, P-168, P-240andP-315, through Agrobacterium rhizogenes receptor-mediated transformed experiment，wetransformed the P-45, P-168, P-240genes into Robinia pseudoacacia，finally observing itseffect on plants’ growth and nodule formation.The results showed that the nodulation process,plant phenotype and nodule development of the infection of P-168, P-240experimental group transgenic Robinia pseudoacacia compared with empty vector control group were not veryobvious. However those plants infected with P-45showed significantly enhancednodulation.Paraffin sections results showed that the nodules internal structure of P-45, P-168and P-240have no significant differences with the control nodule.To study the dynamicexpression characteristics of the target gene during the nodule symbiotic，qRT-PCR showed2zheng45expressed in late nodulation,2zheng168and2zheng240were early expression.The construction of four genes RNAi interference vectors BP-139, BP-168, BP-240,BP-315; through Agrobacterium rhizogenes Receptor-mediated transformed experiment, wetransformed pre-built laboratory interference vector BP-190and BP-287genes into Robiniapseudoacacia，finally observing its effect on plants growth,nodule formation and the infectionprocess.The results showed that BP-190inhibits the formation of hairy roots, we speculated itinvolved in starting lateral roots differentiation and the onset of nodule primordium.With thesilencing BP-287, transformed plants showed small plant size, symbiotic nodulation processsignificant delay and weakness, suggesting the gene of2zheng287played an important rolein the regulation of developmental processes in Robinia pseudoacacia nodulation.To study thedynamic expression characteristics of the target genes in the nodule during the symbiotic，qRT-PCR was used to show2zheng190mainly expressed in the early-mid term while2zheng287mainly in the mid-late term.The construction of five sub-cellular localization vectors G-45, G-139, G-168, G-240and G-315. The acting position of genes G-45, G-168and G-190were studied in cells bytransient expression method in onion epidermal cells.The results showed the translationproducts of G-45gene localized in the plasma membrane, while G-168, G-190mainlylocalized in the nucleus.