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

doi:10.13523/j.cb.2103023

China Biotechnology

2021, Vol. 41

Issue (7): 10-21 DOI: 10.13523/j.cb.2103023

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

FENG Zhao(

),LI Jiang-hao,WANG Jia-hua

College of Medical Technology, Shaanxi University of Chinese Medicine, Xianyang 712046, China

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Abstract

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.

Key words： Ribosomal proteins Symbiotic nodulation RNA interference Subcellular localization Robinia pseudoacacia

Received: 15 March 2021 Published: 03 August 2021

ZTFLH:

Q945

Corresponding Authors: Zhao FENG E-mail: fengzhao2018@163.com

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Cite this article:

FENG Zhao,LI Jiang-hao,WANG Jia-hua. Functional Analysis of RpRPL22, a Ribosomal Protein Homologous Gene, in the Symbiotic Nodulation Process of Robinia Pseudoacacia. China Biotechnology, 2021, 41(7): 10-21.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2103023 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I7/10

Table 1 Primers used in this study

Fig.1 The relative expression of RpRPL22 in different tissues in different periods The data are shown as mean ± SEM, and both biology and technology are repeated 3 times

Fig.2 RpRPL22 sequence alignment with RPL22s from other species

Fig.3 Phylogenetic tree of RpRPL22 (indicated with a red filed circle) and its homologs

Fig.4 Results of subcellular localization (a) Subcellular localization in onion cells, the cell wall has been separated (b) Subcellular localization in R. pseudoacacia hairy roots; GFP alone served as a control, bars=50 μm

Fig.5 The relative expression of GUS gene and target gene were detected 15d and 30d after inoculation (a) RT-PCR was used to analyze the expression of GUS gene in the roots of RNAi transformed plants, and 18S rDNA was used as the control for standardization (b) RT-PCR was used to analyze the expression of the target gene in the roots of RNAi transformed plants and control (c) qRT-PCR was used to compare the expression difference of target gene between RNAi transformed roots and control. The data are shown as mean ± SEM, and both biology and technology are repeated 3 times. P<0.05 was significant difference

Fig.6 Comparison of growth and nodule size between interference plants and control (a, b) Growth of RNAi plants and control(i) (c) Nodules phenotype of RNAi plants and control(i), bars = 1 cm (d, e, f,g) The differences of plant height, root length, fresh weight and nodulation number between the RNAi plants and the control(i) were compared statistically.The data are shown as mean ± SEM, n = 40 and both biology and technology are repeated 3 times. P<0.05 was significant difference

Fig.7 The process of rhizobium infecting the roots of RNAi plants was captured under a fluorescence microscope and the infection events were statistically compared under the microscope (a) Root hair curling; (b, c) Infection threads in root hair and cortex respectively; bars: (a, b) = 50 μm; (c) = 20 μm (d, e, f, g) The differences of root hair curling number/plant, ITs number in root hair/plant, ITs number in cortex/plant and nodule primordia number/plant between the RNAi plants and the control(i) were compared statistically.The data are shown as mean ± SEM, n = 40 and both biology and technology are repeated 3 times. P<0.05 was significant difference

Fig.8 Light microscope photos of nodule longitudinal section and transmission electron microscope of single infected cell (a, e) Light microscope and transmission electron microscope images of paraffin sections of control(i) plant nodules (b, f) Light microscope and transmission electron microscope images of paraffin sections of RpRPL22-RNAi plant nodules (c) Magnification of ZIII in a (d) Magnification of ZIII in b (g) Magnification of image e (h) Magnification of image f (i) Real-time PCR analysis was used to compare the expression level of lentihemoglobin gene in RNAi nodules and control(i) nodules The data are shown as mean ± SEM, both biology and technology are repeated 3 times. P<0.05 was significant difference Bars: (a,b)=100 μm; (c,d)=50 μm; (e,f)=2 μm; (g,h)=0.5 μm


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