蒺藜苜蓿膜联蛋白MtANN2基因的表达模式及盐胁迫下的功能分析

膜联蛋白（annexins）是一类进化保守的多基因家族蛋白，它们广泛存在于真核生物中，能通过Ca²⁺与膜磷脂的结合参与胁迫相关的多种生物学过程。早期对膜联蛋白的研究多集中于脊椎动物，对植物膜联蛋白的认识开始于番茄。关于豆科植物尤其是牧草中膜联蛋白的研究还鲜有报道。本研究分析了蒺藜苜蓿膜联蛋白与饲草紫花苜蓿同源蛋白的进化关系，研究了蒺藜苜蓿膜联蛋白基因MtANN2的表达模式，进一步利用拟南芥同源基因的突变体阐明了MtANN2在根系发育和盐胁迫中的功能。RT-qPCR结果显示，MtANN2在根中高丰度表达，且表达水平受NaCl诱导。RNA原位杂交表明MtANN2特异表达于幼苗侧根原基。拟南芥同源基因AtANN2的T-DNA插入突变体植株弱小、侧根数少、根鲜重低，且对盐（100 mmol·L^-1）处理的敏感性显著高于野生型。超表达MtANN2于atann2后转基因植株的侧根数介于野生型与突变体之间，根鲜重接近野生型，表明MtANN2能在一定程度上互补该突变体的表型缺陷。在盐处理下，该转基因株系的发芽率和长势均恢复到类似野生型的水平。以上结果从分子水平上表明，蒺藜苜蓿膜联蛋白MtANN2参与植物根系生长及盐胁迫响应，高水平表达该基因能够改善植物的耐盐性。本研究为紫花苜蓿耐盐分子育种提供了备选基因。

Expression pattern and biological functions of an annexin encoding gene MtANN2 in Medicago truncatula under salt stress

Annexins are a conserved， multi-member protein family. They are present in eukaryotes and participate in diverse stress-related biological processes by binding membrane phospholipids in a Ca²⁺-dependent way. Most studies of annexins have focused on vertebrates although the first plant annexin was reported early in 1989 from tomato （Lycopersicon esculentum）. To date， information on annexins in legumes， particularly forage legumes， is scarce. In this study， the phylogeny of annexins in Arabidopsis thaliana， Medicago truncatula and Medicago sativa， was analyzed. We then focused on investigating the expression pattern and biological functions of MtANN2 in root development and salt response. Quantitative RT-PCR demonstrated that MtANN2 was abundantly expressed in roots and was NaCl-inducible. RNA in situ hybridization showed a predominant expression of MtANN2 in the lateral root primordia. The loss-of-function mutantof AtANN2， the closest homolog of MtANN2 in Arabidopsis， displayed pleiotropic morphological abnormalities including dwarfism with fewer lateral roots and lower fresh weight， and the homozygous atann2 plants were hypersensitive to the salt treatment （100 mmol·L^-1）. The transgenic atann2 plants ectopically expressed MtANN2 were reminiscent of the wild type with similar fresh weight and the lateral root density between atann2 and Col-0. The results indicated that MtANN2 partly rescued the defects of atann2. Under the NaCl treatment， the germination rate and growth of the transgenic lines were similar to those of the wild type. Our molecular results suggest that MtANN2 is involved in root growth and salt response， and that the increase level of MtANN2 improves salt tolerance. Therefore， MtANN2 may serve as a potential target for breeding alfalfa tolerant to high salinity using the molecular approaches.