氮处理对大豆根瘤固氮能力及GmLbs基因表达的影响

doi:10.11983/CBB21037

摘要/Abstract

摘要： 共生根瘤的固氮效率受外界氮素的严格调控。除固氮酶活性外, 豆血红蛋白(Lb)浓度亦是反应固氮能力的重要指标。为明确氮水平对生物固氮作用的影响, 以大豆(Glycine max)为材料, 在低氮(0.53 mmol·L^-1)条件下接种根瘤菌, 30天后再进行高氮(5.3、10、20、30和40 mmol·L^-1)处理7天, 分析Lb浓度、固氮酶活性及类菌体发育状态。结果表明, 随着外界氮浓度的增加, 根瘤由红变绿, 且红色Lb明显减少而绿色Lb急剧增加; 固氮酶活性显著被抑制, 类菌体中侵染细胞数目和面积显著下降, 表明高氮引起Lb形态的改变与固氮能力关系密切。利用生物信息学及公开表达谱等数据进行分析, 发现大豆根瘤中主要含有4个共生Lb基因, 即GmLb1、GmLb2、GmLb3和GmLb4。4个GmLbs亲缘关系很近且位于进化树的同一分支。进一步分析GmLb1-4转录水平对氮的响应, 结果表明, GmLb1-4的表达显著受高氮抑制。研究结果可为揭示氮介导根瘤衰老机制及生物固氮的应用提供依据。

关键词: 生物固氮, GmLbs, 豆血红蛋白, 氮浓度, 大豆

Abstract: The nitrogen fixation efficiency of symbiotic nodules is tightly regulated by external nitrogen (N). In addition to nitrogenase activity, the leghemoglobin (Lb) amount is vital index contributing to N₂fixation. To determine the effect of environmental N level on biological nitrogen fixation, soybean plants were inoculated with rhizobia for 30 d under low N condition (0.53 mmol·L^-1), then transplanted to hydroponic culture solution with relatively higher N concentrations, including 5.3, 10, 20, 30, and 40 mmol·L^-1 for 7 d, respectively. Lb concentration, nitrogenase activity and bacteroid development status were measured. Nodule displayed changed color from red to green with increasing N concentrations. Consistently, the red Lb concentration gradually declined with increased green Lb concentration. Moreover, the nitrogenase activity, infected cell number and area in nodules were all significantly decreased, suggesting that changes in Lb forms caused by excess N are closely associated with nitrogen fixation capacity. Bioinformatics and public expression profile data displayed that four symbiosis-associated Lbs, including GmLb1, GmLb2, GmLb3, and GmLb4 are the major Lb genes in soybean nodulation. These four GmLbs belong to the same clade of phylogenetic tree. Further analysis on the transcripts of GmLb1-4 in response to N showed that the expression levels of GmLb1-4 were all significantly inhibited by high N. This study services as a reference for future studies in understanding the underlying mechanisms of N-triggered nodule senescence as well as BNF application in agriculture.

Key words: biological nitrogen fixation, GmLbs, leghemoglobin, nitrogen concentration, soybean

杜梦柯, 连文婷, 张晓, 李欣欣. 氮处理对大豆根瘤固氮能力及GmLbs基因表达的影响. 植物学报, 2021, 56(4): 391-403.

Mengke Du, Wenting Lian, Xiao Zhang, Xinxin Li. Effects of Nitrogen Application on Nitrogen Fixation Capacity and GmLbs Expression in Soybean. Chinese Bulletin of Botany, 2021, 56(4): 391-403.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21037

https://www.chinbullbotany.com/CN/Y2021/V56/I4/391

图/表 8

表1 用于qRT-PCR分析的基因特异性引物

Table 1 Gene-specific primers used for qRT-PCR analysis

Name	Primer sequence (5'-3')
GmLb1-qF	CCTCGATACTGGAGAAAGCACC
GmLb1-qR	CAAGTGCGGCATCAATCACC
GmLb2-qF	AATGGAACAGTGGTGGCTGA
GmLb2-qR	AGCACTGCTCAATTCGTCAC
GmLb3-qF	CCGCACTTGGTTCTGTTCAT
GmLb3-qR	TGCTGCCAATTCATCGTAGG
GmLb4-qF	GATCTACTATTGCCGTCAA
GmLb4-qR	GCATCGATTATGATTCACA

图1 供氮水平对大豆根瘤豆血红蛋白(Lb)浓度及固氮能力的影响 (A) 根瘤横切图; (B) 豆血红蛋白浓度; (C) 固氮酶活性。实验设置4个生物学重复, 每个重复随机取2或3个根瘤测定豆血红蛋白浓度和固氮酶活性。数据为10个根瘤的平均值±标准误。不同小写字母代表不同供氮水平间差异显著(P<0.05)。Bars=2 mm

Figure 1 Effects of nitrogen supply on leghemoglobin (Lb) concentration and nitrogenase activity of soybean nodules (A) Cross section of nodules; (B) Leghemoglobin concentration; (C) Nitrogenase activity. There were 4 biological replicates, 2 or 3 nodules were randomly harvested for Lb concentration and nitrogenase activity analysis. Data are means±SE from 10 nodules. Different lowercase letters indicate significant differences among different N supply levels (P<0.05). Bars=2 mm

图2 供氮水平对根瘤侵染细胞发育的影响 (A) 甲苯胺蓝染色的根瘤横切面; (B) 侵染细胞数目; (C) 侵染细胞表面积。数据为40个根瘤横切面的平均值±标准误。不同小写字母代表不同供氮水平间差异显著(P<0.05)。Bars=200 µm

Figure 2 Effects of nitrogen supply on the infected cell development in nodules (A) Toluidine blue-stained nodule cross-sections; (B) Number of infected cells; (C) Surface area of infected cells. Data are means±SE from 40 cross of nodules. Different lowercase letters indicate significant differences among different N supply levels (P<0.05). Bars=200 µm

表2 大豆GmLbs/GmHbs基因信息

Table 2 Information of GmLbs/GmHbs genes in soybean

Gene name	Phytozome locus9.1	Phytozome locus12.0	NCBI number	Accession number	Location coordinates (5'-3')
GmLb1	Glyma10g34260	Glyma.10G198800.1	NM_001248494.2	BT092230	42996028-42997092
GmLb2	Glyma10g34280	Glyma.10G199000.1	NM_001358072.1	FK026737	43004554-43005947
GmLb3	Glyma10g34290	Glyma.10G199100.1	NM_001248999.3	BT092268	43009363-43011021
GmLb4	Glyma20g33290	Glyma.20G191200.1	NM_001248319.3	BT092218	42993081-42994203
GmLb5	Glyma10g34275	Glyma.10G198900.1	-	-	43000045-43003378
GmHb1	Glyma11g12960	Glyma.11G121700.1	NM_001255274.2	BT098807	9299437-9300752
GmHb2	Glyma11g12980	Glyma.11G121800.1	NM_001357481.1	BT096529	9303967-9305430

图3 豆血红蛋白功能域和3D结构预测 (A) 保守功能域预测(黄色字体为血红素结合位点, 黑色字体为辅助因子结合位点, 白色字体为多肽结合位点); (B) 蛋白3D结构预测

Figure 3 Prediction of the conserved domain and 3D protein structure of leghemoglobin (A) Prediction of the conserved domains (the yellow font represents the heme binding site, the black font represents the cofactor binding site, and the white font represents polypeptide binding site); (B) Prediction of 3D protein structure

图4 血红蛋白系统进化树利用MEGA 6.1软件中的邻接法构建系统进化树。

Figure 4 Phylogenetic tree of hemoglobin This phylogenetic tree was constructed by the neighbor-joining method in the MEGA 6.1 program.

图5 大豆GmLbs和GmHbs基因表达热图 (A) RNA-seq数据来源于Soybase网站(https://www.soybase.org); (B) RNA-seq数据来自Libault等(2010)。不同来源的RNA-seq数据通过log10 (FPKM+1)计算后进行热图分析。DAF: 开花后天数; HAS: 播种后小时数; SAM: 顶端分生组织

Figure 5 Heatmaps of GmLbs and GmHbs gene expression in soybean (A) The RNA-seq data obtained from Soybase website (https://www.soybase.org); (B) The RNA-seq data obtained from Libault et al. (2010). RNA-seq data of different sources were calculated with log10(FPKM+1) and further used to generate the heatmaps. DAF: Days after flowering; HAS: Hours after sowing; SAM: Shoot apical meristem

图6 GmLb1-4在不同氮浓度下的相对表达量数据为3个生物学重复的平均值±标准误。

Figure 6 Relative expression value of GmLb1-4 in soybean nodules under different nitrogen concentrations Data are means±SE of 3 biological replicates.

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