紫花苜蓿MsMAX2的克隆及功能研究

doi:10.3864/j.issn.0578-1752.sas-210206

摘要/Abstract

摘要： 【背景】分枝是重要的产量构成因子，在紫花苜蓿育种中至关重要。发掘紫花苜蓿分枝相关基因并明确其作用机理，有助于加快紫花苜蓿高产优质育种。MAX2是重要的分枝相关基因，参与多种植物的分枝调控。【目的】通过对紫花苜蓿MsMAX2功能的研究，为建立MsMAX2调控紫花苜蓿分枝发育分子机制奠定基础。【方法】利用同源克隆的方法从紫花苜蓿中获得MsMAX2的基因序列。通过Expy Protparatam、DNAMAN和MEGA-X等软件对MsMAX2进行序列分析并构建系统进化树。采用实时荧光定量PCR（qRT-PCR）方法分析MsMAX2在紫花苜蓿中的组织表达特异性。运用烟草瞬时表达系统确定MsMAX2蛋白的亚细胞定位。利用农杆菌介导的转化方法获得转基因拟南芥以明确MsMAX2的基因功能。用酵母双杂交技术探明与MsMAX2互作的蛋白。【结果】MsMAX2包含长度为2 136 bp的开放阅读框，编码由711个氨基酸构成的蛋白，属于F-box蛋白超家族。系统进化分析表明，MsMAX2及其同源基因的进化与物种的分化高度相似，表明其是一个功能保守的基因。MsMAX2在紫花苜蓿的颈部组织中表达量最高，苗期叶片和授粉当天的花序中表达量较高，根中次之，其他组织表达量相对较低，暗示其在紫花苜蓿多个组织中发挥作用。亚细胞定位试验表明MsMAX2蛋白定位于细胞核中。在拟南芥max2突变体中过表达MsMAX2可互补突变体的多分枝表型。酵母双杂交试验表明MsMAX2与激素受体D14存在依赖于独脚金内酯的相互作用。【结论】获得在紫花苜蓿颈部组织中高水平表达的MsMAX2，其编码的蛋白定位于细胞核中；MsMAX2在拟南芥多分枝突变体max2中过表达时，能够互补突变表型，表明MsMAX2参与植物的分枝调控，其功能保守。

关键词: 紫花苜蓿, MsMAX2, 分枝, 互补试验, 酵母双杂交

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 (qRT-PCR) 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 2136 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

马琳, 温红雨, 王学敏, 高洪文, 庞永珍. 紫花苜蓿MsMAX2的克隆及功能研究[J]. 中国农业科学, 0, (): 1-.

MA Lin, WEN HongYu, WANG XueMin, GAO HongWen, PANG YongZhen. Cloning and Function Analysis of MsMAX2 gene in alfalfa (Medicago sativa L.)[J]. Scientia Agricultura Sinica, 0, (): 1-.

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紫花苜蓿MsMAX2的克隆及功能研究

Cloning and Function Analysis of MsMAX2 gene in alfalfa (Medicago sativa L.)

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