Functional identification of the role of soybean gene GmPP2C28 in the nitrogen-fixation process of Lotus japonicus

doi:10.11686/cyxb2024427

Abstract

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

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.

Figures/Tables 7

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

Fig.1 Bioinformatics analysis of GmPP2C28 protein

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

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

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

Fig.5 Section staining analysis of the transgenic nodules

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

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