Functional Identification Of Several Nodulin Genes Involved In Symbiotic Nitrogen Fixation Of Robinia Pseudoacacia

Legume plants possess the capacity to establish symbiotic interaction with “rhizobia” insoil, and it culminates in the formation of a unique and specialized organ, the nodule. Theestablishment and development of nodule initiate from a series of molecular dialoguesbetween the symbiotic partners. At the same time, a number of host’s nodulin genes wereinvolved in the whole symbiotic process, and which were regulated precisely. Presently, themolecular mechanisms of symbiotic nodulation of woody legume plants have been receivedlittle attentions and rarely reported. In this paper, we set the symbiotic system consist ofRobinia pseudoacacia L. and Mesorhizobium amorphae CCNWGS0123(abbreviated as186)as the research object for analyzing the function of several nodulin genes separatedpreviously.The main research contents and results were as follows:1. We constructed JK974092and JK974102subcellular localization vectorsrespectively, which were transformed into onion epidermal though gene gun to identify theexpression position of them preliminary. From the result, JK974092（325）as a bindingtranscription factor was consistent with its function prediction for the expression offluorescent signal concentrated in the nucleus. JK974102（327），as a ubiquitin homologousgene，the fluorescence signals are distributed in cells widely, little difference from thecontrol.2. Target gene RNAi vector were constructed to investigate the function of the twogenes(325,327) during nodulation． Agrobacterium rhizogenesis-mediated transformationsystem was applied to introduce RNAi vector into Robinia pseudoacacia after induced thehairy roots to form the "composite seedlings". We harvested silencing vector transformedseedlings roots of the experimental group and the empty vector transformed seedlings roots ofthe control group after inoculation6h,12h,24h,36h,48h,72h,4d, and15d seperately.Moreover, silencing transformed seedlings root and the control were obtained at1d,2d,3d,4dunder non-inoculation conditions. Here, quantitative RT-PCR (qRT-PCR) experimentsconfirmed the expression level of JK974092(325) and JK974102(327) of the RNAi-transformed seeds decreased compared to the control at the corresponding time. Theexpression levels of JK974092(325) of RNAi-transformed seeding root and the controlincreased in as early as6h after inoculation, and its expression level reached the highestexpression level at48h after inoculation. However, the expression level of JK974092decreased at the succeeding4d. This suggested that JK974092(325) has been induced in earlystage of nodulation. Fluorescence labeling for186, Electron microscopy observed theformation of infection threads. The result revealed the infection threads of JK974092RNAi-transformed plants failed to develop normal infection threads, among which could notto reach the inner cortical layers. It was speculated that JK974092affected infection processby regulating the development of infection thread. The nodulation condition performed theJK974092–RNAi seedlings had fewer nodules and slow nodule development compared withthe control. The expression level of JK974102(327)-RNAi performed little change afterinoculation4d, but the expression level reached the highest level at15d. This indicates thatJK974102is induced in the interim of nodulation. Abnormal structure of curved root hairscould be noticed in JK974102-RNAi seedlings by microscope, most of them were heliciform,and the nodules of them were small and white ineffective nodules mostly.3. The over-expression vector of JK974092, JK974102, JK974088(190) and JK974182(287) were constructed separately to investigate their function during nodulation. Overexpression vector were introduced into Robinia pseudoacacia L through Agrobacteriumrhizogenesis K599to grow hairy root for forming the "composite seedlings." In terms oftarget genes JK974092and JK974102, we harvested over-expression vector transformedseedlings roots of the experimental group and empty vector transformed seedlings roots of thecontrol after inoculation0h,6h,12h,24h,48h and72h respectively. Moreover, theover-expression transformed seedlings roots of JK974088and JK974182, and the control at10d,20d,30d,40d after inoculation were obtained. Quantitative RT-PCR (qRT-PCR)confirmed the expression levels of target genes in the over-expression vector transformedseeding roots were higher compared to the control roots. Meanwhile, the tendency and resultsof over-expression of JK974092and JK974102were consistent with RNAi. The expressionlevel of JK974088and JK974182reach the maximal level at40d after inoculation, and thatmeans the two genes may exercise function at the late stage of nodulation. The nodules fromover-expression group were bigger compared to the control, and the number of nodulesshowed no difference.