大豆NADPH氧化酶基因<i>GmRbohL</i>在共生结瘤过程中的功能鉴定

doi:10.7668/hbnxb.20194216

摘要/Abstract

摘要：

植物NADPH氧化酶Rbohs是活性氧(ROS)的主要来源,参与植物的生长、发育、抗逆和植物-微生物的互作等多种生理过程。为探索Rbohs在共生固氮中的功能和作用机制,克隆了1个大豆Rbohs基因家族成员GmRbohL,利用分子生物学、细胞生物学和遗传学等方法,分别对基因的表达模式、蛋白质的亚细胞定位及基因的功能进行了研究。结果显示:GmRbohL基因受根瘤菌诱导,在大豆根和根瘤中特异性高表达。亚细胞定位分析发现,该基因编码蛋白GmRbohL是一种膜蛋白。构建了GmRbohL的植物基因沉默(RNAi)载体,利用发根农杆菌K599介导的大豆毛根转化法,得到转基因毛状根。GmRbohL的基因沉默导致转基因毛状根的根瘤数量明显减少,ROS的产生也受到了抑制。根瘤器官发生阶段根瘤菌的侵染事件明显减少,结瘤标志基因的表达水平也随GmRbohL表达量的降低而降低。根瘤组织切片显示,GmRbohL的基因沉默显著减少了根瘤侵染区共生体的数量,根瘤的固氮酶活性也相应降低。以上数据表明,GmRbohL的基因沉默降低了ROS的产生水平,显著抑制了大豆的共生结瘤过程。推测GmRbohL可能在大豆根瘤的器官发生以及固氮功能调控方面发挥重要的正调控作用。

关键词: 大豆, Rbohs, 活性氧, RNAi, 根瘤形成, 共生结瘤

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

柯丹霞, 冯爽, 胡艺涵, 许丹, 王环. 大豆NADPH氧化酶基因GmRbohL在共生结瘤过程中的功能鉴定[J]. 华北农学报, 2023, 38(5): 29-38. doi: 10.7668/hbnxb.20194216.

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.

http://www.hbnxb.net/CN/Y2023/V38/I5/29

图/表 9

表1 基因表达分析与载体构建所用引物

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

图1 GmRbohL基因的qRT-PCR分析 A.GmRbohL基因在大豆不同组织中的表达特征:分别检测GmRbohL基因在接种根瘤菌BXYD3 28 d茎、叶、根和根瘤以及接种42 d花中的相对表达水平。B.GmRbohL基因在接种根瘤菌后的表达特征:分别检测GmRbohL基因在接种后0 h、1 h、3 h、5 h、12 h、1 d、2 d、3 d、6 d、9 d、14 d、21 d和28 d根(R)以及接种后14,21,28,42 d 根瘤(N)中的表达水平。

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.

图2 GmRbohL蛋白的亚细胞定位分析

Fig.2 Subcellular localization analysis of GmRbohL

图3 阳性转基因毛状根的鉴定绿色箭头所示为带有绿色荧光的阳性转基因毛状根;红色箭头所示为不带绿色荧光的阴性毛状根。

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.

图4 GmRbohL-RNAi转基因毛状根的结瘤表型 A-C.分别为空载体对照(RNAi-EV)、GmRbohL-RNAi-1(GmRbohL-i1)和GmRbohL-RNAi-2(GmRbohL-i2)的毛状根和根瘤的代表性图片。D.接种后28 d“复合体”植株的单株平均根瘤数(n=20)。E.qRT-PCR分析对照和转基因毛状根中GmRbohL的转录水平。不同字母表示对照组与处理组间差异显著(P<0.05)。图5-8同。

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.

图5 GmRbohL-RNAi转基因毛状根的超氧化物水平检测

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

图6 转基因毛状根中侵染事件的统计分析根据侵染线(Infection thread,IT)尖端所在的位置划分不同的根瘤菌侵染事件:侵染线到达卷曲的根毛顶端(A)、到达根表皮或皮层细胞(B)、到达根瘤原基(C)和到达成熟的根瘤中(D)。箭头所指即是侵染线尖端到达的位置。E.接种BXYD3-GUS根瘤菌10 d后转基因毛状根的侵染事件统计。

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.

图7 qRT-PCR检测转基因毛状根中GmEnod40、GmNodulin35、GmCalmodulin和GmLb1的转录水平

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

图8 转基因根瘤的切片染色及固氮酶活性分析 A-C.分别为接种根瘤菌28 d空载体对照、GmRbohL-i1和GmRbohL-i2根瘤的横切面甲苯胺蓝染色图片;D-F.分别为A-C的高倍放大图片:ic.侵染细胞,ui.非侵染细胞;G.根瘤横切面单位面积的侵染细胞数目统计;H.接种根瘤菌28 d RNAi-EV、GmRbohL-i1和GmRbohL-i2根瘤的固氮酶活性测定。

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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[40]	Lei M J, Wang Q, Li X L, Chen A M, Luo L, Xie Y J, Li G, Luo D, Mysore K S, Wen J Q, Xie Z P, Staehelin C, Wang Y Z. The small GTPase ROP10 of Medicago truncatula is required for both tip growth of root hairs and nod factor-induced root hair deformation[J]. The Plant Cell, 2015, 27(3):806-822.doi:10.1105/tpc.114.135210. URL
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[43]	Severin A J, Woody J L, Bolon Y T, Joseph B, Diers B W, Farmer A D, Muehlbauer G J, Nelson R T, Grant D, Specht J E, Graham M A, Cannon S B, May G D, Vance C P, Shoemaker R C. RNA-seq atlas of Glycine max:A guide to the soybean transcriptome[J]. BMC Plant Biology, 2010, 10:160.doi:10.1186/1471-2229-10-160. pmid: 20687943

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大豆NADPH氧化酶基因GmRbohL在共生结瘤过程中的功能鉴定

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

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大豆NADPH氧化酶基因GmRbohL在共生结瘤过程中的功能鉴定

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

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