Characterization of the maize sesquiterpene cyclase genes involved in the defense response to insect damage

植物具有多种机制来保护自己免受食草动物的攻击。一个系统，是由食草昆虫攻击诱导的是萜类化合物的生产，可以直接或间接防御食草动物。先前已经表明，玉米倍半萜环化酶基因stc 1，位于9 S，诱导玉米幼苗被甜菜夜蛾（BAW）幼虫觅食。该基因的产物是一种挥发性萜类化合物，最有可能吸引寄生在BAW幼虫上的黄蜂。导致这一提议的初步工作现在已经表明，stc 2，stc 1在6L中的直系同源物，是由不同的昆虫害虫西南玉米螟（SWCB）诱导的。P.I.s提出了参与这种新型诱导型植物防御系统的两个玉米基因的遗传，分子和生化解剖，如下：1。SWCB和其他相关玉米螟对stc 2基因的诱导的表征。这些实验将建立响应于来自SWCB的杀虫剂的stc 2诱导模式以及植物响应是否对该昆虫物种特异。通过从被SWCB觅食的幼苗鞘中分离全长stc 2 cDNA来确定stc 2基因的完整结构。这将使得能够鉴定蛋白质的N-末端，确认转录物的预测内含子-外显子结构，以及在异源系统中表达STC 2酶以用于其活性的生物化学表征。通过从非诱导型SWCB敏感性Ki 3近交系中分离和测序stc 2基因，评估stc 2作为抗虫基因的可能作用。这将确定Ki 3中stc 2的非诱导性的基础，并提供进一步的证据表明，stc 2对应于SWCB抗性的数量性状位点（QTL），该位点非常接近6L.4中stc 2的位置。异源表达的STC 1和STC 2酶催化的萜类化合物产物性质的测定。这些实验将确定由这些酶产生的萜类化合物的性质，并确认STC 1的产物是否是倍半萜类化合物，如早期生化遗传学数据所示。用将推定的叶绿体转运肽融合到GFP的构建体鉴定STC蛋白最可能的亚细胞定位。这将证实在三个测序的stc 1等位基因中的每一个中预测的N-末端靶向序列是否在体内表现为叶绿体转运肽。这项工作结合了遗传学、分子生物学和生物化学来分析玉米植株对害虫的反应。它将成为对应用生物化学和分子遗传学解决植物生物学实际问题感兴趣的学生和研究生研究人员的优秀培训基地。私家侦探和共同私家侦探。是少数民族的成员，他们的实验室里有大量代表性不足的群体。该项目与美国农业的长期改进有关，因为它解决了玉米保护自己免受昆虫攻击的机制。鉴定与抗虫性有关的基因将有助于育种者开发天然抗虫的作物品种。了解主要作物的自然防御机制将允许替代昆虫控制策略，并减少我们对潜在有毒农药的依赖。因此，所研究的问题具有科学、经济和环境意义。

Plants possess a variety of mechanisms to protect themselves from attack by herbivores. One system that is induced upon attack by herbivorous insects is the production of terpenoids that can serve in either direct or indirect defense against the herbivore. It has previously been shown that the maize sesquiterpene cyclase gene stc1, located in 9S, is induced in corn seedlings being foraged by beet armyworm (BAW) larvae. The product of this gene is a volatile terpenoid that most likely serves to attract wasps that parasitize the BAW larvae. Preliminary work leading to this proposal has now shown that stc2, the ortholog of stc1 in 6L, is induced by a different insect pest, the southwestern corn borer (SWCB). The P.I.s propose a genetic, molecular, and biochemical dissection of the two orthologous maize genes involved in this novel type of inducible plant defense system, as follows:1. Characterization of the induction of the stc2 gene by the SWCB and other related corn borers. These experiments will establish the pattern of stc2 induction in response to regurgitant from the SWCB and whether the plant response is specific to that insect species or not.2. Determination of the complete structure of the stc2 gene by isolating a full-length stc2 cDNA from seedling sheaths that have been foraged by SWCB. This will enable identification of the N-terminus of the protein, confirmation of the predicted intron-exon structure of the transcript, and expression of the STC2 enzyme in a heterologous system for biochemical characterization of its activity.3. Assessment of the possible role of stc2 as an insect resistance gene by isolating and sequencing the stc2 gene from the non-inducible, SWCB-susceptible Ki3 inbred line. This would identify the basis of the noninducibility of stc2 in Ki3 and provide further evidence that stc2 corresponds to a quantitative trait locus (QTL) for SWCB resistance that maps very close to the location of stc2 in 6L.4. Determination of the nature of the terpenoid product catalyzed by the heterologously expressed STC1 and STC2 enzymes. These experiments will identify the nature of the terpenoids made by these enzymes and confirm whether the product of STC1 is a sesquiterpenoid, as suggested by the earlier biochemical genetics data.5. Identification of the most likely subcellular localization of the STC proteins with constructs that fuse their putative chloroplast transit peptides to GFP. This will confirm whether the predicted N-terminal targeting sequence in each of the three sequenced stc1 alleles behaves as a chloroplast transit peptide in vivo.The proposed activity will have clear broader impacts. The work combines genetics, molecular biology, and biochemistry to analyze the response of maize plants to insect pests. It will serve as excellent training ground for student and postgraduate researchers interested in applying biochemistry and molecular genetics to address practical problems in plant biology. Both the P.I and co-P.I. are members of minorities and their labs are heavily populated by under-represented groups. The project is relevant to long-term improvement in U.S. agriculture in that it addresses mechanisms by which maize defends itself from insect attack. The identification of genes involved in insect resistance will aid breeders in developing naturally resistant crop species. Understanding natural defense mechanisms in major crops will allow alternative insect control strategies and reduce our dependence on potentially toxic pesticides. Thus, the problem to be investigated is of scientific, economic and environmental significance.