Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (19): 4061-4069.doi: 10.3864/j.issn.0578-1752.2021.19.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Function Analysis of MsMAX2 Gene in Alfalfa (Medicago sativa L.)

MA Lin(),WEN HongYu,WANG XueMin,GAO HongWen,PANG YongZhen()   

  1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2021-03-05 Accepted:2021-04-19 Online:2021-10-01 Published:2021-10-12
  • Contact: YongZhen PANG E-mail:malin@caas.cn;pangyongzhen@caas.cn

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Abstract:

【Background】 Branching is one of the key yield components, which plays an important role in alfalfa (Medicago sativa L.) breeding. Exploring and functional characterization of key branching-related genes are of significance in accelerating breeding of alfalfa with high yield and quality. MAX2 is an important branching-related gene, which is involved in the regulation of branching in several plant species. 【Objective】 Our research on the functional characterization of MsMAX2 in alfalfa will lay a foundation for the molecular mechanism of MsMAX2 in regulating branch development in alfalfa. 【Method】 The gene sequence of MAX2 in alfalfa was isolated by using homologous cloning. Sequence characteristics and phylogenetic tree of MsMAX2 were analyzed by using bioinformatics tools including Expy Protparatam, DNAMAN, and MEGA-X. The real-time quantitative PCR (qPCR) was applied to detect the tissue-specific expression pattern of MsMAX2 in alfalfa. The subcellular localization of the MsMAX2 protein was determined by using transient expression system in tobacco. The biological function of MsMAX2 was clarified by transformation in the Arabidopsis mutant via Agrobacterium-mediated transformation. Proteins interacting with MsMAX2 were determined by using yeast two-hybrid assay. 【Result】The length of MsMAX2 CDS is 2 136 bp, encoding a protein of 711 amino acids, and it belongs to the F-box protein super-family. Phylogenetic analysis showed that the evolution of MAX2 homologs was highly similar to the differentiation of species, indicating that MsMAX2 was a functionally conserved gene. It was showed that MsMAX2 was expressed in the neck at the highest level, followed by in the leaves of seedling, the inflorescences on pollination day and the roots; the expression level of MsMAX2 was relatively low in other tissues, indicating it functions in multiple tissues. Subcellular localization assay showed that the MsMAX2 protein was localized in the nucleus. Complementation assay in Arabidopsis max2 mutant showed that the multi-branch phenotype was recovered by the ectopic expression of MsMAX2. Yeast two hybrid assay demonstrated that the interaction between MsMAX2 and hormone receptor D14 depended on the existence of strigolactones. 【Conclusion】The MsMAX2 was obtained from alfalfa and it was highly expressed in the neck and the encoding MsMAX2 protein was localized in nucleus. When the MsMAX2 was over-expressed in the Arabidopsis max2 mutant, its multi-branch phenotype was recovered, indicating that MsMAX2 regulates branch development in alfalfa plant, and its function was conserved.

Key words: alfalfa, MsMAX2, branching, complementation assay, yeast two hybrid assay

Fig. 1

Alignment of the predicted amino acid sequences of MsMAX2 with AtMAX2 and OsD3 The F-box domain (in orange frame) and leucine-rich repeat (in black lines)"

Fig. 2

Phylogenetic analysis of MAX2 proteins from 13 plant species AtMAX2: Arabidopsis thaliana, NP_565979.1; GmMAX2: Glycine max, XP_003540983.1; RMS4: Pisum sativum, ABD67495.1; CaMAX2: Cicer arietinum, XP_004505491.1; TpMAX2: Trifolium pratense, PNX93440.1; MtMAX2: Medicago truncatula, XP_003607592.1; OsD3: Orzya sativa, NP_001174608.1; BdMAX2: Brachypodium distachyon, XP_003564315.1; TaMAX2: Triticum aestivum, AZS54115.1; ZmMAX2: Zea may, AQL02030.1; SbMAX2: Sorghum bicolor, XP_002436499.1; SiMAX2: Setaria italica, XP_004964817.1. Proteins for dicotyledons are highlighted with red shadow, and monocotyledons with blue shadow"

Fig. 3

Expression levels of MsMAX2 in various tissues 1: Leaf at seedling stage; 2: Root at seedling stage; 3: Neck at seedling stage; 4: Branch at seedling stage; 5: Head at seedling stage; 6: Stem at branching stage; 7: Leaf at branching stage; 8: Head at branching stage; 9: Stem at flowering stage; 10: Leaf at flowering stage; 11: Inflorescence at flowering stage; 12: Capsule at 0 day after pollination; 13: Capsule at 5 day after pollination"

Fig. 4

Subcellular localization of MsMAX2 in epidermal cells of Nicotiana benthamiana"

Fig. 5

Complementation assays of MsMAX2 in the Arabidopsis max2 mutant The phenotypes (A), MsMAX2 and AtMAX2 relatively expression levels (B) and brunch numbers (C) of WT, max2 and max2 transformed with 35S::MsMAX2 constructs"

Fig. 6

Yeast two-hybrid assays of MsMAX2 with AtD14 and AtSMXL7 GR24 is a synthetic substitute of SLs"

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