细胞壁在云南逸生苜蓿耐铝毒中的作用及机理研究

Aluminum (Al) toxicity is one of the major factors limiting alfalfa production in large-scale acid allitic soils in Southern China. However，strong resistance to acid and Al is a lack of germplasm resources, and the regulation mechanism of Alfalfa on acid and Al resistance is still not entirely clear, which make the resistance breeding work progress slowly. There had existed rich wild alfalfa resources with high acid and Al tolerance in Yunnan. Through our research，alfalfa with different Al resistance have been identified and found the mechanism of different tolerance could not been fully attributed to exudation of organic acids. Therefore, escaped alfalfa resources with different Al tolerance are good to use as the experimental materials in our study. Based on the research of adsorption-desorption of Al by cell wall in root tips，our study will focus on the relation between the structure and composition of cell wall in root tips and Al tolerance. Moreover, potentials, cation exchange capacity and PME of cell wall in root tips will be examined. PME will be analyzed in order to explore its function on regulating Al tolerance of cell wall in root tips. The aim of this study is to deeply reveal Al toxicity to alfalfa and the mechanism of Al tolerance，which will provide theoreticl references for the physiological and molecular mechanisms of Al tolerance in cell wall of root tips of escaped alfalfa in Yunnan.

铝毒是我国南方酸性富铝化土壤中制约紫花苜蓿生产的关键因素，但缺乏特异抗酸铝性强的材料，且人们对紫花苜蓿的抗酸铝调节机制仍不完全清楚，导致抗酸铝性苜蓿育种进展缓慢。云南酸性土壤中孕育了丰富的耐铝苜蓿资源。本研究团队前期对云南逸生紫花苜蓿耐铝性进行了大量研究，发现存在铝敏感型和不敏感型材料，且有机酸分泌不能完全解释苜蓿不同基因型间耐铝性差异。因而，本研究拟以前期筛选的铝敏感型和不敏感型云南逸生紫花苜蓿为材料，在研究苜蓿根尖细胞壁对铝吸收特性的基础上，进一步研究铝胁迫下根尖细胞壁结构与组分变化与苜蓿耐铝性的关系，检测铝胁迫下苜蓿根尖细胞壁电势、果胶甲基酯酶（PME）活性变化的规律，分析根尖细胞壁果胶甲基化程度与苜蓿耐铝性的关系，试图探索苜蓿根尖细胞壁果胶甲基酯化程度对其耐铝性的调控机制，以期更深入揭示紫花苜蓿对铝毒害的耐性机理，为根尖细胞壁在苜蓿耐铝毒中的作用及机理的研究提供理论依据。

本研究对8份云南野生和逸生苜蓿材料进行耐铝性评价，并以高抗的No.12和高感的No.21为材料，研究苜蓿根尖及细胞壁对铝毒的响应及调控，以期揭示苜蓿根尖细胞壁在耐铝性中的作用，为南方地区耐酸铝性苜蓿育种提供理论依据。主要结果如下：.1.低浓度（≤100 µmol/L）铝处理对苜蓿的植株生长及根系伸长的抑制作用较弱；当铝胁迫浓度增加至≥200 µmol/L，苜蓿地上、地下部分生物量显著下降（P<0.05）。随胁迫浓度增加苜蓿根尖横切面细胞变化较大。铝胁迫抑制供试材料叶绿素a和叶绿素b的合成，随着铝浓度的增加，各苜蓿材料的叶绿素含量呈降低趋势。同时，低浓度铝处理（≤100 µmol/L）对苜蓿根系活力抑制作用较大，且促进了丙二醛在根尖的积累。随铝胁迫浓度增加，苜蓿根尖氧化酶活性逐渐降低。采用隶属函数法对供试苜蓿材料耐铝性进行评价，综合排序得出：No.12 > No.22 > No.11 > No.18 > No.27 > No.21 > No.5 > No.3。No.12在不同浓度铝胁迫下表现均较好，No.21较差。.2.以No.12和No.21为材料，研究苜蓿根尖及细胞壁对铝胁迫响应。表明，长时间的铝胁迫（＞12h）会抑制苜蓿根的生长，降低根系活力，诱导根尖胼胝质的形成。苏木精染色结果表明，随处理时间增加，苜蓿根尖横切面细胞出现排列紊乱且表皮细胞受损甚至脱落的现象，敏感型苜蓿21号材料表现较为严重。.3.铝含量分析显示，铝积累主要在苜蓿根尖0-5mm处，且根尖细胞壁铝含量明显高于根尖，敏感型铝积累量更高。随铝胁迫时间延长，供试材料根尖细胞壁果胶、纤维素、半纤维素、蛋白含量增加，且敏感型材料细胞壁多糖含量大于耐受型，但蛋白含量低于耐受型。.4.铝胁迫下，苜蓿细胞壁果胶甲酯酶（PME）活性显著高于根尖，且敏感型PME活性高于耐受型，果胶甲基酯化度（PMD）低于耐受型。随胁迫时间的增加，供试材料根尖细胞壁SOD、POD酶活性及H2O2含量显著上升（P<0.05）。且敏感型材料酶活性、H2O2含量高于耐受型。果胶含量降低，PMD提高，苜蓿根尖细胞壁结合铝的能力变弱，从而提高耐受型苜蓿根系耐受力，增强抗铝性。