Exploring barley mutants for molecular dissection of chlorophyll biosynthetic enzymes and chloroplast development

突变研究在90年前成为一个热门话题。由于大麦叶绿素突变体在苗期表型已经很明显，因此可以作为突变体研究的工具；不能产生叶绿素的突变体很容易被发现为黄色或白色植株，这表明诱变剂处理成功。这些叶绿素突变体一直保存至今，为揭示叶绿素生物合成和叶绿体发育的分子机制提供了一个绝佳的机会。这些突变是致命的，因为没有叶绿素的植物无法存活，但由于大麦种子富含能量，缺乏叶绿素的突变体无论如何都能长到10厘米高的幼苗，这使得对拟南芥突变体无法进行的植物分析成为可能。本研究的长期目标是揭示参与叶绿素生物合成并影响叶绿体发育的相关基因，加深对相应酶和蛋白质的认识。因此，在汉森实验室中，不同的子项目在基因鉴定到蛋白质表征的尺度上处于不同的阶段，我将参与不同阶段的项目。我将进行基因鉴定，利用Xantha-a和Xantha-m突变体在DNA水平上鉴定完全未知的基因xantha和Xantha-m，并通过基因分型测序进行分析。将分别从15个和5个可用的等位突变体中对位于鉴定区域的候选基因进行测序。突变系中有突变的基因表明正确的基因已经被确定。该基因将被克隆并用于大肠杆菌的表达系统，以产生黄原a和黄原m蛋白，用于生化分析和进一步表征。大麦的镁螯合酶和环化酶基因先前已经被汉森实验室的成员鉴定出来，并且已经开发了表达系统。因此，对这些酶的研究更有针对性地了解它们的机制。由于蛋白质的设计揭示了它们的大部分功能，汉森实验室一直专注于使用x射线晶体学和低温电子显微镜进行3D结构研究。我很高兴能参加这些实验，因为这将扩大我的分子技术的曲目。研究叶绿素生物合成的分子基础，特别是大麦中蛋白质水平的叶绿素生物合成的计划，完全符合我未来的科学事业目标。我想建立自己的研究小组，研究温带谷类作物，尤其是小麦的植物结构工程。通过加入最好的大学之一的一个小组，我将获得在几个项目中与硕士和本科生一起工作和指导的经验，并在隆德生物系教授汉森教授的课程中与本科生互动。这将有助于我建立自己的团队，并很快获得教授职位。

Mutation research became a hot topic exactly 90 years ago. Barley chlorophyll mutants were used as a tool in the mutant studies since their phenotype is obvious already at the seedling state; mutants not able to make chlorophyll were easily spotted as yellow or white plants, which indicated a successful mutagen treatment. These chlorophyll mutants have been kept ever since and provide today a fantastic opportunity to reveal the molecular mechanism of chlorophyll biosynthesis and chloroplast development. The mutations are lethal since the plants cannot survive without chlorophyll but due to the energy rich seeds of barley the chlorophyll-less mutants can anyway grow to a seedling of 10 cm, which allow analyses of the plants that cannot be performed with Arabidopsis mutants. The long-term goal of this study is to reveal the genes and deepen the understanding of the corresponding enzymes and proteins participating in chlorophyll biosynthesis and affecting chloroplast development. Thus, in the Hansson lab, different subprojects are at different stages along a gene-identification-to-protein-characterization scale and I will participate in projects at different stages. I will perform gene identification to identify the totally unknown genes Xantha-a and Xantha-m at the DNA level using xantha-a and xantha-m mutants which will be analyzed by genotyping-by-sequencing. Candidate genes located in the identified region will be sequenced from the 15 and 5, respectively, available allelic mutants. A gene with mutations in the mutant lines suggests that the correct gene has been identified. The gene will be cloned and used in expression systems of Escherichia coli in order to produce Xantha-a and Xantha-m proteins for biochemical assays and further characterization. Barley genes of Mg-chelatase and cyclase have previously been identified by members of the Hansson lab and expression systems have been developed. Thus, the studies of these enzymes are more directed to understand their mechanisms. Since the design of the proteins reveal much of their functions, the Hansson lab have focused on 3D structural studies employing X-ray crystallography and cryo-electron microscopy. I am very excited to take part in these experiments since that will broaden my repertoire of molecular techniques. The plan to work with the molecular basis of chlorophyll biosynthesis especially on protein level in barley is perfectly in line with my future scientific career aims. I would like to establish my own research group, performing research towards engineering a plant architecture for temperate cereal crops, especially wheat. From joining a group in one of the best universities, I will gain experience in work with and supervise master and bachelor students in several of the projects, and also interact with undergraduate students at the courses given by Prof. Hansson at the Department of Biology, Lund. This will help in establishing my own group and getting professor ship soon.