关键词: 酸铝胁迫, MATE, 紫花苜蓿, 基因表达调控

Abstract:

【Objective】 Acid and aluminum stress is one of the major environmental stresses in agricultural production in the south of China. Studying the mechanism of native plants adaptation to acid and aluminum stress, and identifying and making use of the excellent gene resources would benefit to breed new varieties of acid and aluminum resistant crops, that will play a fundamental role in molecular breeding of alfalfa. 【Method】 In previous study, full length of BjMATE was cloned from Pingba bitter rape, a native Brassica juncea resource in Guizhou province, and the overexpression vector was constructed by the alfalfa genetic improvement group of Guizhou Institute of Prataculture. The transgenic plants of alfalfa and Arabidopsis thaliana were obtained by tissue culture and flower dipping methods, respectively. Molecular examination was carried out with NPT resistance gene primers and gene-specific primers; and the expression of BjMATE in transgenic plants was examined by qRT-PCR. The germination rate and seedling growth of transgenic and control alfalfa lines were analyzed by hydroponic method under acid, aluminum and acid aluminum combination stress conditions. The alteration of the seedling and root growth was also analyzed between transgenic and Zhongmu 1# alfalfa under a long term but weaker acid-aluminum stress condition. Antioxidant enzyme activity, including POD, SOD, CAT, and MDA concentration alterations between OEMs-5 and Medicago sativa Zhongmu 1# were analyzed with microplate reader under acid, aluminum and acid-aluminum combined stress conditions. At the same time, expression changes of AtMATE, AtPIN2, AtALS3, AtALMT1, and AtSTOP1 which were key genes involving in acid aluminum regulation pathways, were analyzed in the model plant Arabidopsis thaliana by qRT-PCR, and potential regulation network were discussed. 【Result】 The positive rates of transgenic Arabidopsis and alfalfa were 100% and 66.7%, respectively. Compared with the control lines, the expression levels of the candidate transgenic alfalfa and Arabidopsis lines were up-regulated by 63.02 and 76.87 times, respectively. There was no significant difference in seed germination rate between OEMs-5 and Zhongmu 1# under normal, acid stress, aluminum ion stress and acid-aluminum combination stress conditions. While the germination potential was significantly different among the treatments, as well as the materials, OEMs-5 was significantly better than Zhongmu 1#. After a long-term weak stress treatment, the plant height of OEMs-5 was not significantly different from that of Zhongmu 1#; but the biomass and root length of OEMs-5 was significantly better than that of Zhongmu 1# under aluminum stress and acid-aluminum combined stress conditions. Antioxidant enzyme activity and MDA concentration alterations between OEMs-5 and Zhongmu 1# were different. POD and SOD activity increased in both OEMs-5 and Zhongmu 1# after stress treatment, however, significant difference was only detected in SOD assay. No significant difference in CAT activity was detected between the two materials, and the treatments. The content of MDA decreased slightly, significant differences were detected between OEMs-5 and Zhongmu 1# under aluminum stress and acid-aluminum combined stress conditions. Expressions of AtMATE, AtPIN2, AtALS3, AtALMT1 and AtSTOP1 were up-regulated under aluminum stress and acid-aluminum combined stress in both OEMs-5 and Zhongmu 1#, however, no significant difference were detected between them except AtSTOP1.【Conclusion】BjMATE can positively regulate alfalfa tolerance to acid-aluminum stress during seed germination and seedling growth stages. Reactive oxygen species (ROS) scavenger system, especially the activity of SOD, and AtSTOP1 gene which is a key gene involving in acid-aluminum stress regulation pathway, may participate in BjMATE mediated acid-aluminum stress regulation.

Key words: acid aluminum stress, MATE, alfalfa, gene expression regulation