Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (17): 3335-3349.doi: 10.3864/j.issn.0578-1752.2024.17.003

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

Cloning and Biological Function Verification of Alfalfa MsSPL17

CHEN FeiEr(), ZHANG ZhiPeng, JIANG QingXue, MA Lin, WANG XueMin()   

  1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2024-03-27 Accepted:2024-05-11 Online:2024-09-01 Published:2024-09-04
  • Contact: WANG XueMin

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Abstract:

【Objective】Branching is a significant factor influencing alfalfa yield, and the SPL family of transcription factors represents a crucial class of regulatory genes involved in branching (tillering) development in a diverse range of plants. The objective of this reserch is to investigate the biological function of MsSPL17 in alfalfa and to elucidate the role of MsSPL17 in regulating the development of alfalfa meristems. This will provide a valuable reference for alfalfa high-yield biological breeding.【Method】Bioinformatics methods were used to anlyze MsSPL17 sequences and also constructing a phylogenetic tree. The tissue expression specificity of MsSPL17 in alfalfa was analyzed by real-time fluorescence quantitative PCR (qPCR). The subcellular localization of MsSPL17 protein was determined by tobacco transient expression system, and the transcriptional self-activation activity of MsSPL17 was verified. The transgenic alfalfa was obtained by Agrobacterium-mediated transformation and phenotypic analysis was carried out. Transcriptome analysis was utilized to screen for differentially expressed genes in transgenic lines and validate them for further research.【Result】MsSPL17 contained an open reading frame of 1 011 bp, encoding a protein composed of 366 amino acids, belonging to the SBP protein family. Phylogenetic analysis showed that the evolution of MsSPL17 and its homologous genes was highly similar to the differentiation of species, indicating that it is a functional conserved gene. MsSPL17 expressed in all tissues, including stems, nodes, leaves and tops during the critical period of alfalfa growth and development, implied the necessary regulating function of this gene in alfalfa branching. Subcellular localization assay showed that MsSPL17 protein was localized in the nucleus. Transcriptional self-activation assay showed that MsSPL17 did not have self-activation activity and could be used in interacting proteins screening. MsSPL17 transgenic silenced lines exhibited a notable phenotype, including an increase in branch number and stem node number, a reduction in internode length, and an enhancement in nutritional quality.【Conclusion】MsSPL17 was successfully cloned, it expressed in key tissues of alfalfa branching development. The protein encoded by MsSPL17 was localized in the nucleus and demonstrated no transcriptional self-activation activity. Transgenic lines exhibiting multi-branching traits were obtained, and the number of branches increased significantly in yield, while the crude protein content increased in quality.

Key words: alfalfa, SPL gene, branching development, transcriptome, RNAi silencing

Fig. 1

Analysis of MsSPL17 protein sequence A: Prediction of protein conserved domain; B: Prediction of the secondary structure; C: Protein sequences alignment of MsSPL17 with different plant species; D: Protein tertiary structure prediction; E: MsSPL17 was aligned with protein sequences of different species. XP_003625236.2: Medicago truncatula; XP_003625236.2: Arabidopsis thaliana; NP_181749.1: Glycine max; KAH1258171.1: Pisum sativum; XP_050911094.1: Trifolium repens; WJX78628.1: Trifolium pratense; XP_045793008.1: Vicia sepium; XP_027350088.1: Arachis hypogaea; XP_025663422.1: Abrus precatorius; XP_058738332.1: Oryza sativa; XP_015610961.1: Sorghum bicolor; NP_001295407.1: Triticum aestivum; XP_002462571.1: Zea mays"

Fig. 2

Tissue expression pattern of MsSPL17 gene in alfalfa"

Fig. 3

Subcellular localization of MsSPL17 protein"

Fig. 4

Autoactivation test of MsSPL17"

Fig. 5

Identification of alfalfa over-expressing MsSPL17 *** indicates highly significant difference with P<0.001. The same as below"

Fig. 6

Morphological observation of transgenic alfalfa"

Fig. 7

Phenotype data of wild type and MsSPL17 transgenic alfalfa ns: No significant difference; *: Significant difference with P<0.05; **: Highly significant difference with P<0.01"

Fig. 8

Transcriptome data of differential expression genes between groups A: The opposite expression difference Venn diagram of transgenic plants; B: 18 differentially expressed gene heat map"

Fig. 9

Validation by qRT-PCR and RNA-Seq"

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