Isolation And Identification Of Nodule-related Genes Involved In Symbiotic Nitrogen Fixation Of Robinia Pseudoacacia

Legume-rhizobium symbiosis is a complex process involving the special expression of the genes from symbiotic both sides. Many nodulin genes from legumes have been identified, which are expressed specially or upregulatedly during different stages of nodulation. The function of some genes have been clarified. But there are not system studies about the nodulation mechanism of woody legumes. Robinia pseudoacacia, a kind of woody legumes, can form nodule with rhizobia from different genus. However, the genes involved in symbiotic nitrogen fixation of Robinia pseudoacacia are not still reported. Nitrogen (N) starvation is a prerequisite for the formation, development, and function of root nodules, and high levels of combined N in the form of nitrate or ammonium can completely abolish nodule formation. Little is known about the expression of genes related with nodulation in the presence of different combined N source. Therefore, the analysis of genes related nodulation at transcriptional level is necessary to ravel the symbiotic molecular mechanisms in black locust.We researched the spatio-temporal expression of genes related nodulation in symbiotic nitrogen fixation of Robinia pseudoacacia-Mesorhizobium amorphae CCNWGS0123(186) by RT-PCR and qRT-PCR.In the presence of different combined N source, such as N-free,10mM KNO3,20mM KNO3,10mM NH4NO3and10mM AS, the expression of these genes have been studied.The main results were summarized as follows:1. Through RT-PCR and qRT-PCR analysis, ten genes from reverse SSH library which have lower expression levels in the infected roots at30days after inoculation (30r) and the nodules at30days after inoculation (30n) have been confirmed. They are f107, f102, fan37, fan84, fan60, fan141, fanlll, f41,1f10and lf40. The spatio-temporal expression analysis revealed that the transcriptions of8genes could been detected at every stage of nodule development (1f10and lf40not been researched). Generally, the expressions of genes (except fan84) in the roots were reduced at6hour post-inoculation compared with the same age of seedling root without inoculation (4day old, as0). Then there are different degree of rising or falling. fan84、f102were highly expressed at3hour,3day post-inoculation, respectively. We refer fan84and f102function at the recognition between the host plant and symbiotic companion. The rest genes were mainly expressed at the formation of root nodule primordium and the development of nodule. Otherwise, the expressions of8genes were inhibited in nodules.2. In the presence of different combined N source, such as N-free,10mM KNO3,20mM KNO3, lOmM NH4NO3and lOmM AS, the expressions of genes were detected in the6-day-old,10-day-old roots without inoculation or1day after inoculation by qRT-PCR. Whether inoculation or not, the expression levels of genes were higher in the6-day-old roots planted at the presence of combined N than the roots at N-free. Corresponding non-infected roots (6day old), the transcription levels of the genes were reduced in the infected roots planted at the presence of combined N. We Speculate that the expressions of8genes are restrained post inoculation. By the large, the expressions of8genes were impacted differently by the combined N sources and the effects to one and the same gene varied from nitrate and ammonium and their concentrations.