Center for Maize Targeted Mutagenesis

玉米定向诱变中心

• 批准号: 9872644
• 负责人: Robert Martienssen
• 金额: $ 184.22万
• 依托单位: Cold Spring Harbor Laboratory
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-11-01 至 2001-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9872644&HistoricalAwards=false
• 关键词: Center; Maize; Targeted; Mutagenesis

The genetic makeup of crop plants is the fundamental basis for yield, grain quality, plant breeding and crop improvement. In principle, this genetic makeup can be determined by sequencing plant DNA, and in this way identifying most if not all of the genes encoded by a given plant genome. Sophisticated software tools are becoming available that allow a great deal of information to be extracted from sequence data, but ultimately the function of a gene can only be determined by genetic analysis. Classically, this has been achieved by subjecting plants to mutagenizing agents, such as chemicals or X-rays, and then searching for those mutants that have a desired property or trait. The gene mutated in each case can then be identified by a laborious genetic procedure to ultimately isolate the corresponding sequence of DNA. The availability of large amounts of methodically determined DNA sequence information, however, allows a novel, systematic approach to be taken to determine gene function. This approach, first pioneered in model animal genomes such as the fruitfly Drosophila and the nematode worm, C. elegans, involves first constructing a library of thousands of organisms, each of which has a different spectrum of mutations. This library is then screened directly with a DNA sequence to identify mutations in a given gene. Two technological advances make this possible. The first is the use of transposable elements as the mutational agent. Transposable elements were first discovered in maize at Cold Spring Harbor Laboratory by Dr. Barbara McClintock in the 1940s. These elements have a conserved DNA structure enabling them to be readily identified in the genome. They disrupt genes at random, simultaneously providing the mutation of interest, and labeling the gene with the conserved DNA sequence. The second technology is the polymerase chain reaction (PCR), which allows such DNA sequences to be amplified from individual plants giving immediate access to a plant carrying a given mutation. Scientists at Cold Spring Harbor Laboratory have combined these advances to develop a systematic method for determining gene function in maize. Robertson's Mutator transposable elements, first characterized at the DNA level at the University of California at Berkeley, are uniquely suited for this purpose, and a sophisticated genetic strategy has been developed allowing large populations of plants to be screened for mutations in pools. In partnership with Novartis AG, geneticists at Cold Spring Harbor and Berkeley are developing a population of 40,000 plants, and DNA samples are being extracted. Seed from each plant will be cataloged and stored. The PCR reaction will be performed on DNA pools derived from this population so that seed corresponding to mutations in a given gene can be readily identified. Individual geneticists from the maize community will be able to send in sequence information for targeted disruption of genes of interest to them. The information will be entered into a database, and seed corresponding to the mutation will be distributed to interested researchers to allow them to determine the function of each gene chosen in this way. The Mutator Targeted Mutagenesis (MTM) system can thus be used to build up a database of gene function in maize, eventually comprising a significant proportion of the genetic makeup of this crucial crop plant.

作物的遗传组成是作物产量、品质、育种和改良的基础。 原则上，这种基因组成可以通过对植物DNA进行测序来确定，并以这种方式识别由给定植物基因组编码的大多数（如果不是全部）基因。 先进的软件工具可以从序列数据中提取大量信息，但最终只能通过遗传分析来确定基因的功能。 传统上，这已经通过使植物经受诱变剂（例如化学品或X射线），然后寻找具有所需性质或性状的那些突变体来实现。 每种情况下突变的基因都可以通过费力的遗传程序进行鉴定，最终分离出相应的DNA序列。 然而，大量的DNA序列信息的可利用性，允许采取一种新的，系统的方法来确定基因功能。 这种方法首先在模式动物基因组中被采用，如果蝇和线虫C。线虫的基因组学，首先需要构建一个包含数千种生物的文库，每种生物都有不同的突变谱。 然后用DNA序列直接筛选该文库以鉴定给定基因中的突变。 两项技术进步使这成为可能。 第一种是使用转座因子作为突变剂。 转座因子最早是在20世纪40年代由芭芭拉·麦克林托克博士在冷泉港实验室在玉米中发现的。 这些元件具有保守的DNA结构，使得它们能够在基因组中容易地被识别。 它们随机破坏基因，同时提供感兴趣的突变，并用保守的DNA序列标记基因。 第二种技术是聚合酶链式反应（PCR），它允许从单个植物中扩增这种DNA序列，从而立即获得携带特定突变的植物。 冷泉港实验室的科学家们结合这些进展，开发了一种确定玉米基因功能的系统方法。 罗伯逊的突变体转座因子，首先在DNA水平上的特点，在加州大学伯克利分校，是唯一适合于这一目的，和一个复杂的遗传策略已经开发出来，允许大群体的植物进行筛选突变池。 冷泉港和伯克利的遗传学家与诺华公司合作，正在培育4万株植物，并提取DNA样本。 每种植物的种子都将被分类和储存。 将对来源于该群体的DNA池进行PCR反应，以便可以容易地鉴定与给定基因中的突变相对应的种子。 来自玉米社区的遗传学家将能够发送序列信息，以便有针对性地破坏他们感兴趣的基因。 这些信息将被输入数据库，对应于突变的种子将被分发给感兴趣的研究人员，让他们确定以这种方式选择的每个基因的功能。 因此，突变体靶向诱变（MTM）系统可用于建立玉米基因功能的数据库，最终包括这种关键作物植物的大部分遗传组成。