Molecular Cloning of Phycobilisome Polypeptides

植物生物学中的一个主要问题涉及光在叶绿体基因表达调控中的作用，以及叶绿体基因正负调控的机制还不完全清楚。单细胞视紫藻蓝藻属具有许多特性，使其成为研究光调控叶绿体基因的良好系统。这种生物体中的捕光天线由藻胆体组成，藻胆体是含有别藻蓝蛋白、藻蓝蛋白和几个连接物多肽的大分子组件。在黑暗中生长的细胞含有一个小的黄体，它在光下发育成一个大的叶绿体。伴随而来的是APC、CPC和其他叶绿体基因，如psaA、PSBA和rbcL的mRNAs水平增加了50倍。叶绿体光基因的周转率将在黑暗和光照下测定，以确定光中转录产物的积累是由于转录活性增加，还是由于mRNA稳定性的变化，还是两者兼而有之。此外，在黑暗中，血红素促进某些叶绿体光基因转录本的积累，这一过程的机制将被研究(目标1)。将进行实验，以确定连接多肽的基因是否受光调节，以及这些基因的转录水平是否受血红素的调节(目标2)。将对别藻蓝蛋白和藻蓝蛋白的基因进行克隆和测序，以确定可能的光和血红素反应调节元件(目标3)。将开发一个同源的体外转录系统来研究参与叶绿体光基因调控的因素。

A major question in plant biology concerns the role of light in regulation of chloroplast gene expression and the mechanisms for positive and negative control of chloroplast genes are incompletely understood. The unicellular rhodophyte, Cyanidium caldarium, has a number of properties which make it a good system for investigating photoregulated chloroplast genes. The light harvesting antenna in this organism is comprised of phycobilisomes, which are macromolecular assemblies containing allophycocyanin, phycocyanin and several linker polypeptides. Cells grown in the dark contain a small etioplast which develops into a large chloroplast in the light. This is accompanied by 50 fold increases in the levels of mRNAs for apc, cpc, and other chloroplast genes, such as psaA, psbA and rbcL. Turnover rates of chloroplast photogenes will be determined in dark and light to determine whether accumulation of transcripts in light results from increased transcription activity, changes in mRNA stability, or both. In addition, heme promotes accumulation of transcripts for certain chloroplast photogenes in the dark and mechanisms underlying this process will be investigated (Aim 1). Experiments will be performed to determine whether genes for linker polypeptides are photoregulated and whether levels of transcripts of these genes are modulated by heme (Aim 2). Genes for allophycocyanin and phycocyanin will be cloned and sequenced in order to identify possible light- and heme-responsive regulatory elements (Aim 3). A homologous in vitro transcription system will be developed to investigate factors involved in regulation of chloroplast photogenes.