USE OF SYNCHROTRON X-RAY FLUORESCENCE MICROTOMOGRAPHY TO CHARACTERIZE THE GENES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我们的研究结合了现有的基因组技术，如基因缺失和过表达，以及同步辐射X射线荧光(SXRF)显微光谱和显微断层摄影术，以表征参与植物金属离子动态平衡的基因的功能。我们首次将SXRF技术应用于种子液泡铁(Fe)转运相关基因的研究，这对富铁籽粒的发育和人类缺铁性贫血的减少具有深远的意义。虽然成熟种子是SXRF分析的理想样本，但种子表达的基因只是植物基因组的一个子集。为了表征根、叶和发育中的种子中金属(Loid)转运蛋白的编码基因，新鲜或活的植物成像是首选的，因为脱水、树脂渗透和切片可能导致元素和分布人工产物。对活体或新鲜植物成像的限制包括探测器的灵敏度、步进电机的运动以及样品干燥的一系列问题。我们已经在BL2-3进行了初步研究，使我们能够在大约一个小时内从2天大的拟南芥幼苗的切除茎中收集铁和其他重要元素的断层图像，而在其他环境微探测器光束线上进行分析可能需要10个小时。通过比较野生型断层图和缺乏液泡铁转运蛋白(VIT1)的突变体的断层图，我们显示了明显的分布对比，这为该基因的功能提供了新的线索。我们希望在BL2-3进行新鲜和活体植物成像研究，以帮助确定涉及金属离子动态平衡的基因。我们目前的项目包括进一步确定编码液泡铁转运蛋白VIT1的基因，以及负责植物内和种子中砷运输的转运蛋白。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our research combines established genomic techniques, such as gene deletion and overexpression, with synchrotron x-ray fluorescence (SXRF) microspectroscopy and microtomography to characterize the function of genes involved in metal ion homeostasis in plants. We pioneered the use of SXRF in gene characterization with a gene critical for vacuolar iron (Fe) transport in seed, which has far reaching implications for the development of Fe enriched grain and the reduction in Fe deficiency anemia in humans. While mature seed represents an ideal sample for SXRF analysis, seed-expressed genes are only a subset of the plant genome. To characterize genes that encode for metal(loid) transporters in roots, leaves and developing seed, fresh or live plant imaging is preferred, due to the elemental and distributional artifacts that can result from dehydration, resin infiltration and sectioning. Constraints on live or fresh plant imaging range from the sensitivity of the detector, the motion of the stepping motors, as well as a range of issues with sample drying. We have conducted preliminary studies at BL2-3 that have allowed us to collect tomograms of Fe and other important elements from the excised stem of a 2 day old Arabidopsis seedling in approximately one hour, analysis that would have taken 10 hours at other environmental microprobe beamlines. By comparing the wild type tomogram with that of a mutant lacking a vacuolar iron transporter (VIT1) we have shown clear distributional contrasts that have shed new light on the function of the gene. We wish to conduct fresh- and live-plant imaging studies at BL2-3 to help characterize genes involved in metal ion homeostasis. Our current projects involve further characterizing genes that encode for vacuolar Fe transporter, VIT1, and transporters responsible for the transport of arsenic within the plant and into the seed.