中国农业科学2024，Vol.57Issue(17)：3335-3349,中插22-中插23,17.DOI:10.3864/j.issn.0578-1752.2024.17.003
紫花苜蓿MsSPL17的克隆及生物学功能验证
Cloning and Biological Function Verification of Alfalfa MsSPL17
摘要
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.关键词
紫花苜蓿/SPL基因/分枝发育/转录组/RNAi沉默Key words
alfalfa/SPL gene/branching development/transcriptome/RNAi silencing引用本文复制引用
陈菲儿,张志鹏,蒋庆雪,马琳,王学敏..紫花苜蓿MsSPL17的克隆及生物学功能验证[J].中国农业科学,2024,57(17):3335-3349,中插22-中插23,17.基金项目
财政部和农业农村部:国家现代农业产业技术体系(CARS-34)、国家科技资源共享服务平台作物种质资源库(NICGR-63) （CARS-34）
