Cloning of the MtBMI1 gene from Medicago truncatula and its role in drought tolerance

doi:10.11686/cyxb2024303

Abstract

Abstract:

Drought significantly impedes the normal growth， development， and reproduction of plants， and has emerged as a pivotal factor contributing to the decline in crop and forage yields. The BMI1 protein， a key component of the Polycomb Group （PcG） protein complex that mediates histone ubiquitination， is crucial for the epigenetic regulation of plant responses to abiotic stresses. This study reports the cloning of MtBMI1， which encodes a member of the PcG family， from Medicago truncatula. Our analyses showed that the MtBMI1 gene sequence spans 5386 base pairs and encodes a protein comprising 429 amino acids， featuring two functional domains： zf-C3HC4 and RAWUL. Phylogenetic analyses revealed close relationships between MtBMI1 and PvBMI1-1 of Phaseolus vulgaris and GmBMI1-1 of Glycine max. A subcellular localization analysis in tobacco epidermal cells confirmed the nuclear localization of the MtBMI1 protein. Moreover， GUS staining analyses revealed robust activity of the MtBMI1 promoter in the mature inflorescence， stigma， flower， stem， and pedicel of Arabidopsis. Functional analysis via overexpression in Arabidopsis demonstrated that the histone H2AK119ub content was significantly higher （P<0.05） in transgenic lines than in wild type. Under drought stress， transgenic lines exhibited pronounced water-loss phenotypes， with reduced root length， root fresh weight， and aboveground fresh weight （P<0.05）， and elevated levels of malondialdehyde （P<0.05）， compared with the wild type. These findings suggest that MtBMI1 exerts a negative regulatory effect on M. truncatula’s response to drought stress. The results of this study offer insights into the epigenetic mechanisms underlying drought stress tolerance in this model legume species.

Key words: Medicago truncatula, MtBMI1, gene cloning, drought resistance

Yuan-yuan ZHAO, Xiao-jian PU, Cheng-ti XU, Wei WANG, Yun-jie FU. Cloning of the MtBMI1 gene from Medicago truncatula and its role in drought tolerance[J]. Acta Prataculturae Sinica, 2025, 34(6): 139-153.

Figures/Tables 19

Table 1 Summary of primers used for MtBMI1 gene cloning， vector construction and qRT-PCR

引物Primer	引物序列Primer sequence （5'-3'）
Mt-Actin F	TACCCCATTGAGCACGGTAT
Mt-Actin R	ATACATGGCAGGCACATTGA
Mt-BMI1 F	AAGGATGGAAGCGTACCTGTCTCA
Mt-BMI1 R	TGAATCAGCCACAGCTCAACCAA
MtBMI1 F	ATGTCGAATGATGTTGTGAAAGTGA
MtBMI1 R	TCAAGGGCGTGGAGATTTCCG
MtBMI1 MF	TGACCATGGTATCGAATGATGTTGTGAAAGTGA
MtBMI1 MR	AGGGTGACCTCAAGGGCGTGGAGATTTCCG
pC3301-R	AGTAACATAGATGACACCGC
MtBMI1 promoter F	TGTATCGAGAGGTGTGTAATCAGTAC
MtBMI1 promoter R	CACTTTCACAACATCATTCGACAT
MtBMI1 promoter MF	TACCCGGGGATCCTCTAGAGTATCGAGAGGTGTGTAATC
MtBMI1 promoter MR	ACCCTCAGATCTACCATGGATATTCTTGATGATTTCTTGCT
MtBMI1 GFP F	TGGAGAGGACACGCTCGAGATGTCGAATGATGTTGTGAA
MtBMI1 GFP R	CCCTTGCTCACCATGAATTCAGGGCGTGGAGATTTCCG
Bar F	ATGAGCCCAGAACGACGCCC
Bar R	TCAAATCTCGGTGACGGGCA

Fig.1 The characteristics of MtBMI1 gene

Fig.2 The full length of CDS and corresponding amino acid sequence of MtBMI1

Fig.3 Conserved domains， secondary and tertiary structures of MtBMI1 protein

Fig.4 Multiple sequence alignment of BMI1 protein

Fig.5 Phylogenetic tree construction of BMI1 gene

Fig.6 Expression profiles of MtBMI1 in different developmental stages and tissues of M. truncatula

Fig.7 Expression profiles of MtBMI1 under different concentrations of mannitol

Fig.8 Subcellular localization of MtBMI1 gene in tobaccoleaf

Fig.9 MtBMI1 promoter cloned by RT-PCR

Fig.10 GUS staining results of pCAMBIA3301-MtBMI1 promotertransgenic Arabidopsis

Fig.11 MtBMI1 gene cloned by RT-PCR

Fig.12 The relative expression levels of MtBMI1 in transgenic Arabidopsis lines

Fig.13 Phenotypic comparison of transgenic and wild-type Arabidopsis at seedling and bolting stages

Fig.14 Phenotypic comparison of transgenic and wild-type Arabidopsis under 300 mmol·L-1 mannitol treatment

Fig.15 Effects of mannitol treatment on the content of malondialdehyde and proline in transgenic and wild-type Arabidopsis

Fig.16 Evaluation of drought resistance in MtBMI1 transgenic Arabidopsis

Fig.17 Analysis of H2AK119ub histone content in MtBMI1 transgenic Arabidopsis

Fig.18 Expression levels of epigenetic modification related genes in transgenic Arabidopsis

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