Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (21): 4544-4556.doi: 10.3864/j.issn.0578-1752.2025.21.021

• EXPLORATION OF SALT-ALKALI AND DROUGHT RESISTANT GENES FOR ALFALFA BREEDING • Previous Articles    

Phosphate Transporter MsPT5 Regulates Phosphate Uptake and Utilization in Alfalfa

HUANG HongMei1,2(), WANG SiQi1,2(), YANG QingChuan2, GUO ChangHong1,*(), WANG Xue2,*()   

  1. 1 Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province/College of Life Science and Technology, Harbin Normal University, Harbin 150025
    2 Beijing Institute of Animal Science, Chinese Academic of Agriculture Science, Beijing 100193
  • Received:2024-12-10 Accepted:2025-03-28 Online:2025-11-01 Published:2025-11-06
  • Contact: GUO ChangHong, WANG Xue

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Abstract:

【Objective】This study aimed to explore the function of MsPT5, a phosphate transporter in alfalfa, so as to provide the theoretical basis and genetic resources for analyzing the response of alfalfa to low phosphate stress and cultivating phosphate nutrition efficient alfalfa. 【Method】Alfalfa was subjected to both full phosphate and low phosphate stress treatments, and root systems were selected for transcriptome sequencing. Weighted gene correlation network analysis was used to screen for the key gene MsPT5 in response to low phosphate stress in alfalfa; the experiment was carried out to clone the CDS sequence of MsPT5 gene, and the transmembrane domain of MsPT5 protein was analyzed using DNAMAN and TMHMM online websites; Super1300: GFP plant expression vector was used as the backbone, and the overexpression vector Super1300:MsPT5-GFP was constructed through enzyme digestion and homologous recombination; the recombinant vector Super1300:MsPT5-GFP was transformed into Agrobacterium GV3101 using the freeze-thaw method, and Agrobacterium was injected into tobacco for subcellular localization observation; Super1300:MsPT5-GFP was transferred into Arabidopsis through Agrobacterium mediated inflorescence infection, and homozygous strains were screened for inorganic phosphate content determination, biomass statistics, and arsenate phenotype detection; the tissue culture method was employed to obtain MsPT5 transgenic alfalfa, and the positive plants were screened for biomass statistics, phosphate content, and protein content determination. 【Result】MsPT5 was a member of the PHT1 family, with 12 transmembrane domains located on the cytoplasmic membrane; the physiological indicators of Arabidopsis thaliana showed that the inorganic phosphate content of MsPT5 overexpressing strains was 23.02 and 22.30 nmol·mg-1, respectively, while the inorganic phosphate content of the wild-type control was 19.97 nmol·mg-1. The biomass of MsPT5 overexpressing strains were 0.047 g/plant and 0.054 g/plant, respectively, while the biomass of the wild-type control was 0.026 g/plant. Overexpression of MsPT5 could increase the inorganic phosphate content and biomass of Arabidopsis; the results of arsenate phenotype detection showed that the MsPT5 overexpression material exhibited a significant arsenic toxicity phenotype; the physiological indicators of alfalfa showed that the biomass of MsPT5 transgenic alfalfa was 19.39 g/plant, 18.62 g/plant, and 16.65 g/plant, respectively, while the biomass of the wild-type control was 15.14 g/plant. The phosphate content of MsPT5 transgenic alfalfa was 0.37%, 0.39%, and 0.38%, respectively, while the wild-type control had a phosphate content of 0.30%. The protein content of MsPT5 transgenic alfalfa was 21.37%, 21.54%, and 19.91%, respectively, while the protein content of the wild-type control was 18.04%. The biomass of MsPT5 transgenic alfalfa was significantly increased, while the phosphate and protein content were significantly higher than the control. 【Conclusion】 MsPT5 was an important gene in alfalfa's response to low phosphate stress. Overexpression of MsPT5 could increase plant phosphate absorption capacity and phosphate content, and improve alfalfa yield and quality. Therefore, MsPT5 was important for cultivating high-yield and high-quality alfalfa.

Key words: alfalfa, MsPT5, phosphate transport, phosphate content, biomass

Table 1

Primer sequence used in the study"

引物名称
Primer name		 引物序列
Primer sequence (5′→3′)		 用途
Usage
MsPT5-super1300-F
MsPT5-super1300-R		 tctagaaagcttctgcaggggATGTCAGGAGAGCTAGGAGTGC
gctcctcgcccttgctcaccatAACCCTTGATCCTTCTT GCTCA		 克隆
Gene cloning
MsActin-F
MsActin-R		 CAAAAGATGGCAGATGCTGAGGAT
CATGCACCAGTATGACGAGGTCG		 紫花苜蓿qRT-PCR内参基因
Alfalfa qRT-PCR internal reference gene
MsPT5-RNA-F
MsPT5-RNA-R		 TGATGTGGCCAAGACACAAT
CTGGTCTTGTCGGATTTGGT		 RNA水平鉴定
RNA level identification
MsPT5-DNA-F
MsPT5-DNA-R		 GTCGTTTATTTCGGCGTGTA
CAAAGCAACAATTCCACCAC		 DNA水平鉴定
DNA level identification
AtActin-F
AtActin-R		 GAAATCACAGCACTTGCACC
AAGCCTTTGATCTTGAGAGC		 拟南芥qRT-PCR内参基因
Arabidopsis qRT-PCR internal reference gene

Table 2

Differentially expressed genes screened under low phosphate stress"

比较组
Comparison		 所有差异基因数量
All DEGs		 上调基因
Up-regulated DEGs		 下调基因
Down-regulated DEGs
root_P0 vs root_P 5829 3504 2325

Fig. 1

WGCNA analysis A: Hierarcgical cluster tree illustrating the modules identified by WGCNA; B: Module eigengene expression pattern; C:Heat map analysis of PHT1 family phosphate transporters; D: Evolutionary analysis of PHT1 family members in Medicago sativa and Medicago truncatula"

Fig. 2

Transmembrane structure prediction of MsPT5"

Fig. 3

Protein sequences alignment of PHT1 family members"

Fig. 4

Subcellular localization of MsPT5"

Fig. 5

Identification of transgenic Arabidopsis A: Identification of MsPT5 gene in Arabidopsis; B: Relative expression of MsPT2 in Arabidopsis"

Fig. 6

Physiological indexes of Arabidopsis different genotypes A: Pi content in different Arabidopsis genotypes; B: Biomass of different Arabidopsis genotypes"

Fig. 7

Arsenate tolerance phenotype analysis A: Arsenate tolerance phenotype analysis; B: Green cotyledon rates calculation"

Fig. 8

Physiological indexes measurement of MsPT5-overexpression alfalfa A: Relative expression of MsPT5 in alfalfa; B: Phenotypic detection of MsPT5-overexpression alfalfa; C: Biomass of alfalfa different genotypes; D: P contents of alfalfa different genotypes; E: Protein contents of alfalfa different genotypes"

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