EAGER: Quantitative Analysis of Alternative Splicing in Plants Using Surface Enhanced Raman Spectroscopy

PI: Burkhard Schulz（普渡大学）CoPI: Joseph Irudayaraj（普渡大学）本项目开发并测试了一种检测和定量植物中选择性剪接基因的新方法。高等生物中的大多数基因由外显子和内含子组成，内含子阻断了基因的蛋白质编码序列。然而，在转录过程中，这些内含子并不总是精确地剪接并从前体RNA上移除。这导致来自同一基因的不同mRNA变体。它极大地增加了表达蛋白质的可变性和数量，并解释了为什么细胞中的蛋白质比基因多数千种。选择性剪接（AS）在植物发育过程中起着重要的调控作用。为了更深入地了解这一过程的作用，在细胞水平上对不同剪接变异体进行精确的量化是必要的。这个项目是一个创新的方法来检测和定量几个AS产生的mRNA变异在一个单一的实验。这是一种检测mRNA变异的高灵敏度方法。该项目结合了基于纳米技术的表面增强拉曼散射（SERS）和新型纳米材料的使用。SERS利用金纳米粒子增强激光束的拉曼散射信号，这些分子（dna、蛋白质等）在这些增强信号的金纳米粒子的纳米距离内。该项目的目标是：(1)开发用于检测植物mRNA剪接变异体的纳米技术工具箱；(2)评估AS定量的检出限；(3)开发基于sers的定量策略，用于在亚器官水平上检测小细胞数量的剪接变异。这个跨学科的项目是植物遗传学家和生物物理学家之间的密切合作，以定量分析植物细胞中的mRNA变异。它将为纳米技术方法在更广泛的植物科学领域的应用奠定基础，其影响将超越基础研究。该项目将为工程和植物科学专业的学生创造新的机会，将他们的教育组合扩展到传统课程之外的领域，为他们的职业发展开辟新的途径。计划在初高中学生的研究夏令营期间开展面向公众的外展活动。将建立一个发布详细协议和其他背景资料的参考网站，可通过http://www.hort.purdue.edu/hort/people/faculty/schulz.shtml访问。它将提供有关资源、新开发的纳米材料和教育材料的信息，以培养纳米技术方面的学生和博士后研究人员。利用纳米技术分析植物中砷化砷的方案和程序也将通过在适当期刊上的出版物和在科学会议上的通讯提供给科学界。

PI: Burkhard Schulz (Purdue University)CoPI: Joseph Irudayaraj (Purdue University)This project develops and tests a novel method for the detection and quantification of alternatively spliced genes in plants. Most genes in higher organisms are structured into exons and introns, the latter interrupting the protein coding sequence of the genes. Yet these introns are not always precisely spliced and removed from a precursor RNA during transcription. This results in different mRNA variants from the same gene. It greatly increases the variability and number of expressed proteins and explains why there are thousands more proteins than genes in a cell. It is becoming clear that this process of alternative splicing (AS) plays an important role in regulating developmental processes in plants. A precise quantification of different splice variants, also on the cellular level, is necessary in order to gain a deeper understanding of the role of this process. This project is an innovative approach to the detection and quantification of several AS generated mRNA variants in a single experiment. It is a highly sensitive method for detecting mRNA variants. This project combines nanotechnology based Surface Enhanced Raman Scattering (SERS) and the use of novel nanomaterials. SERS uses gold nanoparticles to enhance the Raman scattering signal of a laser beam by molecules (DNAs, proteins, etc) within nanometer distance of these signal-enhancing gold nanoparticles. The objectives of the project are: (1) to develop a nanotechnology toolbox for detection of mRNA splicing variants in plants; (2) to assess detection limits for quantification of AS; and, (3) to develop a SERS-based quantification strategy for the detection of splicing variants in small cell numbers on a sub-organ level.This interdisciplinary project is a close collaboration between plant geneticists and biophysicists to quantitatively profile mRNA variants in plant cells. It will generate a foundation for the application of nanotechnology approaches in the broader field of plant sciences with impacts beyond basic research. The project will create new opportunities for students in engineering as well as plant sciences that will expand their educational portfolio in fields not part of their traditional curricula, opening new avenues for their career development. Outreach activities to the public and during research summer camps for middle school and high school students are projected. A reference website that publishes detailed protocols and additional background information will be established with access through http://www.hort.purdue.edu/hort/people/faculty/schulz.shtml. It will provide information on resources, newly developed nanomaterials and educational material to train students and postdoctoral researchers in nanotechnology. Protocols and procedures to analyze AS in plants using nanotechnology will also be made available to the scientific community through publications in appropriate journals and communications at scientific conferences.