Exploring calcium homeostasis and signalling in Aspergillus nidulans by using a calcium auxotrophy and a calcium-responsive reporter

钙是大多数（如果不是所有）生命形式的必需营养素。例如，在哺乳动物中，它的作用包括肌肉收缩、神经系统的正常运作、细胞分裂能力、受精以及牙齿和骨骼的形成。在其在植物中的作用中，它对温度变化的反应和水的可用性以及使大气中的氮气被纳入生长和维持生命所需的有机分子的共生关系至关重要。在细菌和真菌中，钙在生物体的结构和各种形式（如孢子）的形成中起着重要作用。它也可能在细菌和真菌感染动物和植物的能力中发挥作用。通过更多地了解钙在真菌中的各种作用，我们不仅增加了对一种普遍存在的重要生物群体的基本知识，而且还潜在地确定了我们可以阻止真菌生长的方法。例如，如果我们能确定钙在真菌中发挥的作用，但在人类中不起作用，那么就有可能找到治疗人类真菌感染的药物。这类药物有相当大的需求，因为目前存在的药物很少，而且由于真菌的突变，这些药物可能会失去效力。同样，确定钙在真菌中而不是在植物中所起的作用，有助于设计出能够保护农业或园艺重要植物的杀菌剂。它还可能指出防止真菌破坏食品和其他商品的新方法。这一提议涉及到使用一种土壤传播的真菌，即对动物和植物几乎没有致病性的细粒曲霉，但它非常容易使用传统的遗传方法和现代的基因修饰方法进行操作。在这项工作中，我获得了两个相当独特的工具，它们将被证明在确定环境和真菌之间钙分布的因素、真菌细胞内钙的分布及其在真菌中的作用方面非常有用。其中之一是突变的组合，导致需要添加钙来获得真菌的正常生长。正常情况下，生长所需的钙量非常小，以至于钙污染了水和其他生长所需的化学物质就足够了。这种需要钙的生物的行为本身就令人感兴趣，但除此之外，它可以通过DNA损伤剂（诱变剂）处理和寻找不再需要添加钙来生长的衍生生物来获得影响钙反应的进一步突变。另一种工具涉及一种基因，这种基因可以在某种化学物质存在的情况下产生蓝色。这个基因的反应，取决于是否形成蓝色，如果形成，颜色有多深，受添加的钙和未满足的钙需求的影响。通过用诱变剂处理含有该基因的生物体，我们可以获得在添加钙或不添加钙的情况下，在蓝色形成或强度上与亲本不同的突变体。最后，我们将使用一种将水母基因插入真菌的系统。这种基因合成的蛋白质对钙的反应是发出蓝光。这为我们提供了一种方法，可以跟踪钙对影响其在突变活生物体中的行为的处理的行为，这些突变活生物体对钙的反应是由上述两种工具中的一种或两种工具决定的。

Calcium is an essential nutrient for most, if not all forms of life. For example, among its roles in mammals, it is essential for muscle contraction, the proper functioning of the nervous system, the ability of cells to divide, fertilisation and the formation of teeth and bones. Among its roles in plants, it is essential for responses to temperature changes and the availability of water and the symbiotic relationship which enables nitrogen gas in the atmosphere to be incorporated into organic molecules needed for growth and the maintenance of life. In bacteria and fungi calcium plays roles in the structure of the organisms and in formation of various forms such as spores. It is also likely to play a role in the ability of bacteria and fungi to infect animals and plants. By learning more about the various roles of calcium in fungi we not only add to basic knowledge about an ubiquitous and important group of organisms but potentially identify ways in which we can prevent fungal growth. For example, if we can identify roles that calcium plays in fungi but does not play in humans then potentially drugs can be found that can treat fungal infections in humans. There is a considerable need for drugs of this sort because very few currently exist and there is a danger because of mutant fungi that these might lose their effectiveness. Similarly, identifying roles that calcium plays in fungi but not in plants can aid in the design of fungicides able to protect plants of agricultural or horticultural importance. It might also point to new ways of preventing spoilage of foods and other commodities by fungi. This proposal involves the use of a soil-borne fungus, Aspergillus nidulans, which is virtually non-pathogenic, to both animals and plants, but which is very easy to manipulate using both traditional genetic methods and modern genetic modification methods. For this work I have obtained two rather unique tools which should prove extremely useful in identifying factors responsible for the distribution of calcium between the environment and the fungus, its distribution within fungal cells and its roles within the fungus. One of these is a combination of mutations which results in a requirement for adding calcium to obtain normal growth of the fungus. Normally, the amount of calcium required for growth is so tiny that calcium contamination of water and the other chemicals required for growth suffices. The behaviour of this calcium-requiring organism is of interest in its own right but, in addition, it can be used to obtain further mutations affecting calcium responses by treating with a DNA damaging agent (mutagen) and seeking derivative organisms which no longer require addition of calcium for growth. The other tool involves a gene made in such a way that a blue colour can be produced in the presence of a certain chemical. The response of this gene, as determined by whether a blue colour is formed and, if so, how intense it is, is influenced by added calcium and by an unfulfilled calcium requirement. By treating an organism containing this gene with a mutagen, we can obtain mutants which differ from their parent in blue colour formation or its intensity in the presence or absence of added calcium. Finally, we will use a system in which a jellyfish gene is inserted into the fungus. The protein made from this gene responds to calcium by emitting blue light. This provides us with a means of following the behaviour of calcium in response to treatments which affect its behaviour in living mutant organisms whose response to calcium is altered as determined by one or both of the above two tools.