RUI: Molecular Genetic Analysis of Chlorophyll b Synthesis in Arabidopsis

在维管植物和非维管植物、绿藻和原叶绿藻中，叶绿素(Chl)b结合并稳定光系统II的捕光复合蛋白。Chl b是由Chl a在一个需要叶绿素a加氧酶的反应中合成的，该加氧酶是拟南芥AtCAO基因的产物。该项目将产生过表达AtCAO的转基因植株，以观察Chla和Chlb的稳定比例是否会受到干扰。将产生含有构成启动子和可诱导启动子的植物，测量AtCAO的mRNA和蛋白质水平，并测定Chl b的合成速率。将对高产品系的表型进行检查，以确定是否形成了超大的PSII捕光复合体。第二个目标是生产一种用于测量蛋白质水平和通过免疫金标记进行亚细胞器定位的AtCAO抗体。第三个目标涉及一种突变的条件氯离子，它在中等光强下降低了Chl，特别是Chlb的水平。这条线在中等光强下呈现黄绿色，这种可见的表型与位于内、外天线复合体的LHCb蛋白的部分丢失有关。这条线已经被用来筛选在中等光强下变得不那么黄绿的突变体，CCH抑制子。这些抑制系中的一个已经被证明具有在F2分离期间可见的表型。对这一基因以及其他抑制基因进行了表征和定位。最终目标是在实验室启动一个新的项目；分析核Elip1和Elip2基因的非常强光响应启动子。在非常高的光照强度下，两个Elip基因在四个小时后都被诱导了100倍。这些研究可能有助于理解驱散激子的叶绿体与细胞核通讯所需的因素。随着对Chl b合成的调节和定位以及叶绿体-核通讯的进一步了解，有才华的本科生和硕士学位学生，其中许多来自科学界代表性较低的少数族裔，将接受研究生涯的培训。

Chlorophyll (Chl) b binds and stabilizes the light harvesting complex proteins of photosystem II in vascular and non-vascular plants, green algae and prochlorophytes. Chl b is synthesized from Chl a in a reaction that requires chlorophyll a oxygenase, the product of the Arabidopsis AtCAO gene. This project will yield transgenic plants that overexpress AtCAO in order to observe if the stable ratio of Chl a to Chl b can be perturbed. Plants with constitutive and inducible promoters will be produced, AtCAO mRNA and protein level will be measured, and rates of Chl b synthesis will be determined. The phenotypes of overproducing lines will be examined to determine if an extra large PSII light harvesting complex is formed. The second objective will be the production of an AtCAO antibody for measurement of protein levels and for suborganelle localization by immunogold labeling. The third objective concerns a mutant, conditional chlorina, that has decreased levels of Chl, especially Chl b, under moderate light intensity. This line appears yellow-green under moderate light intensity, and this visible phenotype correlates to partial loss of Lhcb proteins located in the inner and outer antenna complex. This line has been used to screen for mutants that turn less yellow-green under moderate light intensity, cch suppressors. One of these suppressor lines has been shown to have a phenotype that is visible during segregation of the F2. Characterization and mapping of this, and other suppressors, are proposed. The final objective is to initiate a new project in the laboratory; an analysis of the very high-light responsive promoters of the nuclear Elip1 and Elip2 genes. Both Elip genes are induced 100-fold after four hours at very high light intensity. These studies could lead to an understanding of the factors that are required for the exciton-dissipating chloroplast to communicate with the nucleus. Along with furthering knowledge about the regulation and localization of Chl b synthesis, and chloroplast-nuclear communication, talented undergraduate and Masters degree students, many of them from minorities that are underrepresented in the sciences, will receive training for research careers.