刺槐核糖体蛋白同源基因RpRPL22在共生结瘤过程中功能研究

目的: 核糖体蛋白(RPs)属于多功能蛋白,能够参与调控细胞生长和响应胁迫条件。RpRPL22是一个从豆科植物刺槐中分离得到的结瘤相关基因,通过序列比对发现其与核糖体大亚基蛋白RPL22高度同源。对其如何通过调控根瘤菌侵染而在共生结瘤过程中发挥重要作用进行了较为深入的探索。方法: 利用实时荧光定量PCR技术(qRT-PCR)分析RpRPL22在接菌后不同时间及不同植物组织的表达变化。利用cDNA末端快速扩增技术(RACE)获得目的基因cDNA全长。通过GFP报告基因进行RpRPL22亚细胞定位分析。通过Gateway BP重组技术构建RNA干扰(RNAi)重组载体,借助电转化法将重组载体转至农杆菌K599,利用农杆菌介导植物根部,接菌后观察和测量植株表型。首先从宏观水平统计观察目的基因是否对结瘤过程有影响,其次从分子水平揭示目的基因在共生结瘤过程的重要功能。结果: 不同接菌时期、不同植物组织目的基因qRT-PCR相对表达量结果显示,几乎在所有取样的接菌时间,目的基因RpRPL22在接菌根中的相对表达量都低于未接菌对照根,只有接菌后第25天除外。在成熟的根瘤中,接菌后第25天该基因的表达量也最高。洋葱表皮和毛状根亚细胞定位结果均显示在椰菜花叶病毒(CaMV)的35S启动子控制下,RpRPL22融合绿色荧光蛋白GFP的荧光信号在细胞核和细胞质有明显的表达。RNAi转化植株的表型统计观察结果,比如植株鲜重、植株的有效结瘤数目较对照组均有明显的降低;同时RNAi转化植株在根瘤菌侵染过程形成的侵染线数目和根瘤原基数目较对照均显著降低。根瘤切片实验用于观察根瘤显微超微结构,结果显示RNAi植株根瘤中固氮区的受菌侵染细胞数目与对照相比明显减少。电镜观察根瘤单个受菌侵染细胞中类菌体形态显示,RNAi根瘤中类菌体侵染细胞胞体多呈不规则形状,皱缩变形严重,环类菌体周间隙空间增大,多共生体融合,表现出细胞凋亡的迹象。对照根瘤中的受菌侵染细胞胞体多呈圆形椭圆形,胞质饱满丰富且分布均匀,细胞发育正常,表明RNAi植株根瘤发育过程明显受阻。结论: 核糖体蛋白(RP)能够参与调控豆科植物共生结瘤过程,相关同源基因RpRPL22可能在起始根瘤菌侵染植物和阻止类菌体降解过程中起重要作用。

Functional Analysis of RpRPL22, a Ribosomal Protein Homologous Gene,in the Symbiotic Nodulation Process of Robinia Pseudoacacia

Objective: Ribosomal proteins (RPs) have many functions as known, including participation in regulating cell growth and responding to stress conditions. The object of this research is RpRPL22, a nodulation-related gene isolated from the legume Robinia pseudoacacia, which has a high degree of homology with the large ribosomal subunit protein RPL22 through sequence alignment. It has conducted a more in-depth exploration on how RpRPL22 regulates the infection of rhizobia and plays an important role in the process of symbiotic nodulation.Methods: Real-time fluorescence quantitative PCR technology (qRT-PCR) was used to reveal the expression level of RpRPL22 at different time after inoculation and also in different tissues. GFP reporter gene was employed to identify the subcellular localization of RpRPL22. Rapid amplification of the cDNA ends (RACE) technology was applied to get the full-length cDNA of the target gene. The RNA interference (RNAi) recombinant vector was constructed through the Gateway BP recombination technology and then transformed into Agrobacterium K599 through electrotransformation and then infect the radicle. After inoculated with rhizobia, transformed line phenotypes were observed and statistically analyzed to verify RpRPL22 function in the symbiotic nodulation process of R. pseudoacacia. To observe whether the target gene has an influence on the nodulation process from the macro level statistics, and then reveal the important function of the target gene in the symbiotic nodulation process from the molecular level.Results: Gene expression analysis showed that, with the exception of the 25th day post-inoculation (dpi), the expression level of RpRPL22 in the inoculated roots decreased compared with the non-inoculated control. In matured nodules, the RpRPL22 expression was peaked at the 25th dpi. The results of subcellular localization of both onion epidermis and hairy root showed that fluorescence signals of RpRPL22-GFP fusion were distributed in nucleus and cytoplasm under the control of 35S promoter of cauliflower mosaic virus (CaMV). The phenotypic observation results of RNAi transformed plants, such as fresh weight and effective nodulation number, were significantly lower than those of the control group. At the same time, the number of infection lines and nodule primordia was significantly lower in RNAi transformed plants than in the control. Nodule section is used to observe the microscopic and ultrastructure of nodules, which showed that the infected cells in the nitrogen-fixing zone (ZIII) in the nodules of RNAi plants were significantly reduced compared with the control group. Observation of the bacteroid morphology in a single infected cell of the root nodule by transmission electron microscope revealed that the bacteroid in RNAi nodules was severely shrunk and deformed and only a small amount of bacteroids were contained in infected cells. Besides, the fusion of multiple symbiosomes occurred and the space between the bacteroids increased. In contrast, the infected cells in the control nodules developed normally, with smooth edges and uniform cytoplasm. The above indicated that the nodule development process of RpRPL22-RNAi plants was obviously blocked.Conclusion: Ribosomal protein (RP) can participate in the regulation of the symbiotic nodulation process of leguminous plants, and the related homologous gene RpRPL22 may play an important role in initiating rhizobia infecting plants and preventing the degradation of bacteroids.