苜蓿应答干旱和盐胁迫的机理及MtPHD6基因的功能分析

发布时间：2018-03-07 17:17

本文选题：苜蓿　切入点：非生物胁迫　出处：《中国科学院研究生院(武汉植物园)》2016年博士论文　论文类型：学位论文

【摘要】：干旱和盐害是影响植物生长、发育、生产力和分布的两个主要环境因子。水分缺失和土壤盐渍化已经成为可持续发展农业的两大瓶颈。另外,随着人口的急剧增加和环境问题的日益恶化,全球农业产量将很难再满足世界粮食和能源的需求。因此,如何合理地利用干旱和盐渍化土壤是可持续农业急需解决的一个问题。苜蓿是豆科苜蓿属一年生或多年生草本植物,因具有丰富的氨基酸和蛋白质,被誉为“牧草之王”,具有较强的抗旱耐盐能力,在我国滨海盐碱地区和西北干旱半干旱地区都有广泛种植。虽然在轻度胁迫下,苜蓿可以正常种植生长,但重度胁迫条件下,苜蓿的生产力和分布范围受到一定的影响。研究表明不同苜蓿品种的抗旱和耐盐性存在品种间差异。因此,选择抗性较强的苜蓿品种,研究苜蓿抗性相关基因的功能原理,对于培育高产高抗性的苜蓿新品种,扩大苜蓿的种植范围具有重要意义。在本研究中,我们首先以紫花苜蓿(Medicago sativa L.)为研究对象,分别筛选出具有不同抗旱性和耐盐性的两种紫花苜蓿品种,比较分析抗性不同的两种紫花苜蓿品种在相应胁迫条件下形态,生理和分子水平上的差异。其次,以蒺藜苜蓿(Medicago truncatula L)(Jemalong A17)为研究对象,克隆MtPHD6基因,并将其转入到拟南芥,对转基因植株进行不同浓度梯度的甘露醇(Mannitol),盐(NaCl)和脱落酸(ABA)胁迫处理,观察MtPHD6转基因植株对各种胁迫的响应。主要研究结果如下：(1)通过对10种紫花苜蓿品种进行抗旱性的初步筛选,获得抗旱性较强的陇东和干旱相对敏感的阿尔冈金。对筛选出的两品种进行为期18天的干旱处理,研究表明在正常生长条件下,抗旱性强的陇东叶片大小和气孔密度都小于干旱敏感的阿尔冈金,从而使陇东表现出较低的水分失水率。干旱胁迫处理后,与阿尔冈金相比,陇东所受的细胞膜损伤较低,脯氨酸和抗坏血酸含量较高,活性氧(ROS)积累量较少。而且,陇东中抗氧化物酶活力在干旱胁迫条件下显著高于阿尔冈金。另外,在干旱胁迫条件下,陇东叶和根中胁迫相关基因的表达量如MtDehyd,MSNAC, MfCBF4和MsHSP23等显著高于阿尔冈金。上述研究结果表明,陇东品种的抗旱性高于阿尔冈金,最终表现在干旱条件下叶片枯萎较少,侧根数较多,地上／地下单株生物量较大,复水后存活率较高。(2)对14种紫花苜蓿品种进行耐盐性的初步筛选,获得耐盐性较强的211609和盐相对敏感的新疆大叶。其次,对筛选出的两品种进行为期18d的250mM NaCl处理,研究发现在盐胁迫条件下,与新疆大叶相比,211609表现出较高的叶片含水量(LWC)和可溶性蛋白含量,较低的细胞膜损伤和活性氧(ROS)积累量,较高的谷胱甘肽(GSH)含量和抗氧化物酶活力。另外,盐胁迫下,211609中胁迫相关基因,例如NHX1, ZFG, CBF4和HSP23等的表达量显著高于新疆大叶。研究结果表明,211609品种的耐盐性高于新疆大叶品种。(3)通过基因克隆及转化,我们成功获得MtPHD6转基因拟南芥植株,并筛选出表达量相对较高的两个转基因株系MtPHD6-4和MtPHD6-7。在不同浓度的Mannitol, NaCl和ABA处理下,研究结果发现两个转基因株系的萌发率和野生型相比无明显差别,但在高浓度胁迫条件下子叶变绿值均显著高于野生型。综上所述,不同紫花苜蓿品种之间的抗性存在显著差别,通过在胁迫条件下形态,生理和分子水平上的比较分析,研究结果显示陇东品种的抗旱性高于阿尔冈金品种；211609品种的耐盐性高于新疆大叶品种。另外,我们发现MtPHD6转基因拟南芥植株可以更好地在高浓度胁迫平板上生长。上述研究结果希望为今后苜蓿高抗性育种提供一定的科学理论依据。
[Abstract]:Drought and salinity affect plant growth, development, two major environmental factors and productivity distribution. Water loss and soil salinization has become the bottleneck of agricultural sustainable development in two. In addition, with the rapid increase of population and the worsening environmental problems, global agricultural production will be difficult to meet the needs of the world food and energy therefore, how to reasonably use the drought and salinization of soil is a problem of sustainable agriculture need to be resolved. Alfalfa is the legume Medicago annual or perennial herbaceous plants, because of its rich amino acid and protein, known as the "king of grass", with strong salt tolerance and drought resistance ability, are widely planted in China coastal saline areas and arid and semi-arid area of Northwest China. Although under mild stress, alfalfa can normal plant growth, but the severe stress condition, productivity and distribution range of Alfalfa Affected. Research shows that different alfalfa varieties of drought resistance and salt tolerance among different varieties. Therefore, selection of stronger resistance alfalfa varieties, function principle related genes of Alfalfa resistance to new alfalfa varieties, breeding high yield and high resistance, it is of great significance to expand the scope of alfalfa planting. In this study, we begin with alfalfa (Medicago sativa L.) as the research object, were screened with different drought resistance and salt tolerance of two alfalfa varieties, comparative analysis of different resistance of two alfalfa varieties in the corresponding stress conditions, differences in physiological and molecular level. Secondly, in Medicago truncatula (Medicago truncatula L) (Jemalong A17) as the research object, the cloning of the MtPHD6 gene, and transformed into Arabidopsis thaliana, mannitol of different concentration gradient of transgenic plants (Mannitol), salt (NaCl) and removal Abscisic acid (ABA) treatment, observation of MtPHD6 transgenic plants in response to various stresses. The main results are as follows: (1) through the preliminary screening of drought resistance of 10 Alfalfa varieties, drought resistance and drought in obtaining strong relatively sensitive. The Algonquin of two varieties were screened for drought treatment the 18 day, research shows that under the condition of normal growth, strong drought resistance in leaf size and stomatal density are smaller than the gold Algonquin sensitive to drought which showed lower water loss in rate. After drought stress, compared with Al Gen Kim, in the cell membrane damage was lower, and higher proline the content of ascorbic acid, reactive oxygen species (ROS) accumulation was less. Moreover, the antioxidant enzyme activity in Longdong under drought stress was significantly higher than that of Algonquin. In addition, under drought stress, the leaves and roots The expression of stress related genes such as MtDehyd, MSNAC, MfCBF4 and MsHSP23 were significantly higher than that of Algonquin. The results showed that the drought resistance of Gansu were higher than those of the Algonquin, the final performance under drought conditions, leaves wither less lateral root number of ground / underground single biomass larger, higher survival rate after rewatering. (2) to conduct a preliminary screening of salt tolerance of 14 alfalfa varieties, have stronger salt resistance and salt 211609 relatively sensitive Xinjiang Daye. Secondly, the two cultivars were screened for 250mM NaCl 18D, the studies now under salt stress, compared with Xinjiang Daye, 211609 a higher leaf water content (LWC) and soluble protein content, cell membrane damage and lower reactive oxygen species (ROS) accumulation, higher glutathione (GSH) content and antioxidant enzyme activity. In addition, under salt stress, stress in the 211609 phase Close the gene, such as NHX1, ZFG, CBF4 and HSP23 expression was significantly higher than that of Xinjiang Daye. The results show that the salt tolerance of 211609 varieties was higher than that of Xinjiang big leaf species. (3) by gene cloning and transformation, we successfully obtained MtPHD6 transgenic Arabidopsis plants, and screened the expression of relatively high two transgenic plant the Department of MtPHD6-4 and MtPHD6-7. in different concentrations of Mannitol, NaCl and ABA, the results of the study found that the germination rate of two transgenic lines and wild type compared with no significant difference, but in high concentration at the leaves turn green values were significantly higher than that of wild type. In summary, there are significant differences between the resistance of different alfalfa varieties and through the form in stress conditions, comparative analysis of physiological and molecular level. The results showed that drought resistance in varieties higher than Al Gen Kim varieties; salt tolerance variety is higher than 211609 Xinjiang leaf cultivar. In addition, we found that MtPHD6 transgenic Arabidopsis plants could grow well on high concentration stress plates. The above results hope to provide some scientific and theoretical basis for future high resistance breeding of alfalfa.

【学位授予单位】：中国科学院研究生院(武汉植物园)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S541.9

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