大豆GmPP2C28基因对百脉根结瘤固氮的功能研究

doi:10.11686/cyxb2024427

摘要/Abstract

摘要：

植物蛋白磷酸酶PP2C是植物至关重要的一类丝/苏氨酸蛋白磷酸酶。该家族基因在植物发育及多种逆境响应中起着关键作用，关于其在豆科植物与根瘤菌共生结瘤过程中的功能研究相对较少。本研究前期克隆了1个大豆PP2C家族基因GmPP2C28，并证实其受根瘤菌诱导表达。构建GmPP2C28基因的过表达载体p1302G-GmPP2C28，利用发根农杆菌LBA1334介导的百脉根毛根转化法获得带转基因毛状根的百脉根嵌合体植株。通过结瘤试验发现转GmPP2C28基因百脉根结瘤数目明显高于转空载体的对照植株，结瘤指示基因的转录水平显著上调。对根瘤切片进行甲苯胺蓝染色发现，过量表达GmPP2C28基因显著增加根瘤侵染区类菌体的数量。进一步对根瘤的固氮酶活性进行测定发现，过量表达GmPP2C28基因显著增加成熟期及衰老期根瘤的固氮酶活性。以上结果表明在百脉根中异源表达GmPP2C28基因，显著增加了嵌合体百脉根植株结瘤数目以及根瘤中类菌体的数量。此外，过表达GmPP2C28显著提高了成熟期以及衰亡期根瘤的固氮酶活性，从而大大延缓了根瘤的衰老。研究结果可为创制优良百脉根品种，充分发挥百脉根的生物固氮作用提供新的候选基因。

关键词: 大豆, 蛋白磷酸酶2C, 百脉根, 共生固氮, 根瘤衰老

Abstract:

Protein phosphatase 2C （PP2C） is a crucial serine/threonine protein phosphatase in plants. These enzymes are encoded by a large gene family， and they play key roles in plant development and various stress responses. However， there is relatively little research on the role of PP2Cs in the formation of nodules in the symbiotic relationship between leguminous plants and rhizobia. Previously， a soybean （Glycine max） PP2C family gene GmPP2C28 was cloned and its expression was confirmed to be induced by rhizobia. In this study， we constructed a plant overexpression vector p1302G-GmPP2C28 and obtained transgenic Lotus japonicus plants using Agrobacterium tumefaciens LBA1334-mediated transformation of hairy roots. The transgenic GmPP2C28-expressing plants formed significantly more nodules than did those transformed with the empty vector， and the transcript level of the nodule indicator gene also tended to be higher in the transgenic plants than in the empty vector control plants. Methylamine blue staining of nodule slices revealed that overexpression of GmPP2C28 significantly increased the number of bacteroids in the infected area of the nodules. Analyses of nodule nitrogenase activity revealed that overexpression of GmPP2C28 significantly increased nitrogenase activity in mature and senescent nodules. Together， these results show that heterologous expression of GmPP2C28 in L. japonicus significantly increased the numbers of nodules and filamentous bacteria in the root nodules. In addition， overexpression of GmPP2C28 significantly increased nitrogenase activity in mature and decaying nodules， thereby greatly delaying nodule aging. The findings of this study identify a new candidate gene for creating excellent varieties of L. japonicus and other legume species with strong biological nitrogen fixation.

Key words: soybean, protein phosphatase 2C, Lotus japonicus, symbiotic nitrogen fixation, nodule senescence

柯丹霞, 侯仕博, 周兆源, 马云浩, 陈志杰, 宋晓莉, 林佳诺. 大豆GmPP2C28基因对百脉根结瘤固氮的功能研究[J]. 草业学报, 2025, 34(10): 164-173.

Dan-xia KE, Shi-bo HOU, Zhao-yuan ZHOU, Yun-hao MA, Zhi-jie CHEN, Xiao-li SONG, Jia-nuo LIN. Functional identification of the role of soybean gene GmPP2C28 in the nitrogen-fixation process of Lotus japonicus[J]. Acta Prataculturae Sinica, 2025, 34(10): 164-173.

图/表 7

表1 本研究中所使用的引物

Table 1 The primers used in this study

引物名称 Primer name	引物序列 Sequence of primer （5′-3′）
F-OX	GGAATTCATGGGTATCTGC
R-OX	TCCCCCGGGTTAGTTACCAAG
F-GUS	GTCGCGCAAGACTGTAACCA
R-GUS	CGGCGAAATTCCATACCTG
F-NIN-rt	AACTCACTGGAAACAGGTGCTTTC
R-NIN-rt	CTATTGCGGAATGTATTAGCTAGA
F-ENOD40-1-rt	GGAGGTATGCTCAAACATTC
R-ENOD40-1-rt	GTAACTTCTCAAGAGAAGACC
F-ENOD40-2-rt	CAAAACTCGTTATGTTGCGG
R-ENOD40-2-rt	CACCTCAAAGGAAGAAGAACA
F-GmPP2C28-rt	TTGCAACGGTCGTGTATTTGCG
R-GmPP2C28-rt	TGGGTAACCACACTCTCTGGATG
F-UBI	TTCACCTTGTGCTCCGTCTTC
R-UBI	AACAACAGCACACACAGACAATC

图1 GmPP2C28蛋白的生物信息学分析A： GmPP2C28蛋白的保守结构域分析。B： GmPP2C28蛋白的3D结构预测。C： GmPP2C28蛋白的磷酸化位点分析。A： Conservative domain analysis of GmPP2C28 protein； B： 3D structure prediction of GmPP2C28 protein； C： Phosphorylation site analysis of GmPP2C28 protein.

Fig.1 Bioinformatics analysis of GmPP2C28 protein

图2 GmPP2C28基因在不同时期大豆根和根瘤中的表达水平dR代表接种不同天数的根组织。dN代表接种不同天数的根瘤组织。**代表P<0.01。下同。dR represents root tissues inoculated for different days. dN represents nodule tissues inoculated for different days. ** represents P<0.01. The same below.

Fig.2 Expression levels of GmPP2C28 gene in soybean roots and nodules at different stages

图3 嵌合体百脉根阳性毛状根的分子生物学检测A：毛状根根尖的GUS染色图，箭头所示为深蓝色的阳性毛状根。B： PCR检测毛状根中GUS基因的表达。C： RT-PCR检测毛状根中GmPP2C28的表达。多聚泛素（UBI）作为内参基因。Wt：野生型。Ev：空载体对照。M：DNA Marker Ⅲ。Bars=5 mm。A： The GUS staining image of the root tip of the hair root， the dark blue positive hair root indicated by the arrow. B： PCR detection of GUS gene expression in transgenic and control hairy roots. C： RT-PCR was used to detect the expression level of GmPP2C28 in transgenic and control hairy roots. Polyubiquitin as an internal reference gene. Wt： Wild-type. Ev： Empty vector control. M： DNA Marker Ⅲ molecular weight standard.

Fig.3 Molecular biology detection of positive hairy roots in composite L. japonicus

图4 过表达GmPP2C28基因对百脉根结瘤的影响A：接种根瘤菌28 d后嵌合体植株的结瘤表型。B：单株嵌合体植株的平均结瘤数目。C：荧光定量PCR检测嵌合体百脉根毛状根中结瘤指示基因的表达水平。GmPP2C28-OX：超表达GmPP2C28嵌合体植株。Ev：空载体对照。下同。Bars=10 mm。n=15。A： The nodulation phenotype of complex plants after 28 days of inoculation with rhizobia. B： The average number of nodules in a single composite plant. C： Fluorescence quantitative PCR was used to detect the expression levels of nodule indicator genes in the hairy roots of complex L. japonicus. GmPP2C28-OX： Over-expressing GmPP2C28 complex plants. Ev： Empty vector control. The same below.

Fig.4 Effect of overexpression GmPP2C28 gene on nodulation in composite L. japonicus

图5 转基因根瘤的切片染色分析A，B：分别为接种根瘤菌28 d时对照（Ev）和GmPP2C28-OX根瘤的横切面甲苯胺蓝染色图片。C，D：分别为A和B的高倍放大图片。黑框部分为放大位置。ic：侵染细胞。ui：非侵染细胞。E：接种根瘤菌28 d时Ev和GmPP2C28-OX根瘤切片单位面积侵染细胞的数目。*代表 P<0.05。Bars=200 μm （A，B），50 μm（C，D）。n=20。A，B： Cross sectional images of Ev and GmPP2C28-OX nodules stained with toluidine blue after 28 days of inoculation with rhizobia， respectively. C，D： High magnification images of A and B respectively. The black box indicates the enlarged position. ic： Infecting cells. ui： Non invasive cells. E： The number of infected cells per unit area in Ev and GmPP2C28-OX nodule sections after 28 days of inoculation with rhizobia. * represents P<0.05.

Fig.5 Section staining analysis of the transgenic nodules

图6 不同时期转基因根瘤的固氮酶活性测定

Fig.6 Determination of nitrogenase activity in transgenic root nodules at different stages

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