Functional Identification of Soybean NADPH Oxidase Gene GmRbohL in the Nodulation Process

doi:10.7668/hbnxb.20194216

Abstract

Abstract:

The plant NADPH oxidase Rbohs(Respiratory burst oxidase homologs) is the main source of reactive oxygen species (ROS),which participate in various physiological processes such as plant growth,development,stress resistance and plant-microorganism interaction.In order to explore the function and mechanism of Rbohs in symbiotic nitrogen fixation,GmRbohL,a member of soybean Rbohs gene family,was cloned in this study.The gene expression pattern,protein subcellular localization and gene function were studied by molecular biology,cell biology and genetics,respectively.The results revealed that: GmRbohL gene was induced by rhizobia and expressed specifically in soybean roots and nodules.Subcellular localization analysis indicated that the gene-encoded protein GmRbohL was a membrane protein.The plant gene silencing (RNAi) vector of GmRbohL was constructed,and the transgenic hairy roots were obtained by the transformation of soybean hairy root mediated by Agrobacterium rhizogenes K599.Gene silencing of GmRbohL resulted in a significant reduction in the number of nodules of transgenic hairy roots,and the production of ROS was also inhibited. Gene silencing of GmRbohL reduced the infection of rhizobia at the stage of root nodule organogenesis,and the expression level of nodulation marker genes also decreased with the decrease of GmRbohL expression.The root nodule tissue sections showed that gene silencing of GmRbohL significantly reduced the number of symbionts in the infected area of root nodules,and the nitrogenase activity of root nodules also decreased accordingly.The above data indicated that gene silencing of GmRbohL significantly inhibits the symbiotic nodulation process of soybean by reducing the production level of ROS.It is speculated that GmRbohL may play an important positive regulatory role in organogenesis of soybean nodules and regulation of nitrogen fixation function.

Key words: Soybean, Rbohs, ROS, RNAi, Nodule formation, Symbiotic nodulation

KE Danxia, FENG Shuang, HU Yihan, XU Dan, WANG Huan. Functional Identification of Soybean NADPH Oxidase Gene GmRbohL in the Nodulation Process[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 29-38. doi: 10.7668/hbnxb.20194216.

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Figures/Tables 9

Tab.1 Primers used for gene expression analysis and vector construction

引物名称 Primer name	正向引物(5'-3') Forward primer(5'-3')	反向引物(5'-3') Reverse primer(5'-3')	用途 Purpose
GmRbohL-GFP	GGGGTACCATGGTGGAGATCACGCTG	GCTCTAGACTAAAAATTTTCTTTGTG	亚细胞定位
GmRbohL-i1-1	CATGCCATGGCCAACACGAGTCATGGTC	GGCGCGCCCCTCGTCGTCCTTGAAC	RNAi载体构建
GmRbohL-i1-2	AACCCGGGCCAACACGAGTCATGGTC	CCCGGATCCCCTCGTCGTCCTTGAAC
GmRbohL-i2-1	CATGCCATGGGTAGTGGGTGGTATATAG	GGCGCGCCCTGATGATAGTATGAAAG
GmRbohL-i2-2	AACCCGGGGTAGTGGGTGGTATATAG	CCCGGATCCCTGATGATAGTATGAAAG
GmRbohL-qRT	AACCTAGCCACGTATGCACCAC	TCAGTGGTCCGCTCAGAG	qRT-PCR检测
GmActin11-qRT	GAGCTATGAATTGCCTGATGG	CGTTTCATGAATTCCAGTAGC
GmEnod40-qRT	CACTTCCTGATACCCGTGAAA	CGCCACTCAAGAAAGAATGTT
GmNodulin35-qRT	CACTTCCTGATACCCGTGAAA	AAAGTAAAGCGGCTTCTGAGG
GmCalmodulin-qRT	TCTCCCAGTCCAAGATCACC	GCCGATATTTTCCCATCTCC
GmLb1-qRT	CCTCGATACTGGAGAAAGCACC	CAAGTGCGGCATCAATCACC

Fig.1 qRT-PCR analysis of GmRbohL A.GmRbohL gene expression characteristics in various soybean tissues.The relative expression of GmRbohL gene was evaluated in stems,leaves,roots and nodules of plants harvested at 28 days post inoculation(dpi) with BXYD3.Flowers were harvested at 42 dpi with BXYD3.B.GmRbohL gene expression characteristics after rhizobial inoculation.The soybean roots(R)were collected at 0 h,1 h,3 h,5 h,12 h,1 d,2 d,3 d,6 d,9 d,14 d,21 d and 28 d post inoculation.The soybean nodules(N)were collected at 14,21,28 and 42 dpi.

Fig.2 Subcellular localization analysis of GmRbohL

Fig.3 Identification of positive transgenic hairy roots The green arrow indicates the positive transgenic hairy roots with green fluorescence.The red arrow indicates the negative hairy roots without green fluorescence.

Fig.4 Nodulation phenotype of GmRbohL-RNAi transgenic hairy roots A-C.Representative images of hairy roots and nodules from hairy roots expressing empty vector (RNAi-EV),GmRbohL-RNAi-1 (GmRbohL-i1),and GmRbohL-RNAi-2 (GmRbohL-i2) respectively.D.Mean number of nodules per plant of soybean at 28 dpi (n=20).E.qRT-PCR analysis of the transcript level of GmRbohL in the control and transgenic hairy roots.Different letters indicate significant difference between the control group and the treatment groups(P<0.05).The same as Fig.5-8.

Fig.5 Superoxide accumulation in GmRbohL-RNAi transgenic hairy roots

Fig.6 Analysis of infection events in transgenic hairy roots Rhizobial infection events were scored based on the place where the infection thread (IT) tips located at:the top of curled root hairs (A),the root epidermal or cortical cells (B),the nodule primordias (C) and the mature nodules (D).Arrows indicate the location of the IT tips.E.The average number of infection event was scored in transgenic hairy roots at 10 dpi with the BXYD3-GUS strain infection.

Fig.7 qRT-PCR analysis of the transcript levels of GmEnod40,GmNodulin35,GmCalmodulin and GmLb1 in the transgenic hairy roots

Fig.8 Section staining and nitrogenase activity analysis of the transgenic nodules A-C show toluidine blue-stained transverse sections of nodules at 28 dpi from transgenic control,GmRbohL-i1 and GmRbohL-i2 roots;D-F.Higher magnification images of A-C:ic.Infected cells,ui.Uninfected cells;G.Number of infection cells in transverse sections of nodules;H.Nitrogenase activities of nodules of RNAi-EV,GmRbohL-i1 and GmRbohL-i2 at 28 dpi.

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