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

植物有各种各样的机制来保护自己免受食草动物的攻击。在受到食草昆虫的攻击时，诱导的一个系统是产生萜类化合物，可以直接或间接地防御食草昆虫。先前已有研究表明，玉米倍半萜环化酶基因stc1位于9S，在被甜菜粘虫（BAW）幼虫觅食的玉米幼苗中被诱导。这种基因的产物是一种挥发性萜类化合物，最有可能用来吸引寄生在BAW幼虫上的黄蜂。初步研究表明，6L中stc1的同源基因stc2是由一种不同的害虫——西南玉米螟虫（SWCB）诱导的。P.I.s建议对这一新型诱导植物防御系统中涉及的两个同源玉米基因进行遗传、分子和生化解剖，如下：SWCB和其他相关玉米螟虫诱导stc2基因的特性研究。这些实验将确定stc2诱导对SWCB反刍反应的模式，以及植物的反应是否针对该昆虫物种。通过从SWCB采集的幼苗鞘中分离stc2全长cDNA，确定stc2基因的完整结构。这将有助于鉴定蛋白质的n端，确认转录物的内含子-外显子结构，并在异源系统中表达STC2酶以进行其活性的生化表征。通过从非诱导型、swcb敏感的Ki3自交系中分离和测序stc2基因，评估stc2作为抗虫基因的可能作用。这将确定stc2在Ki3中不可诱导性的基础，并进一步证明stc2对应的SWCB抗性数量性状位点（QTL）与stc2在6L.4中的位置非常接近。异源表达STC1和STC2酶催化萜类产物性质的测定。这些实验将确定这些酶产生的萜类化合物的性质，并确认STC1的产物是否如早期生化遗传学数据所提示的是倍半萜类化合物。鉴定最有可能的STC蛋白亚细胞定位与构建融合其假定的叶绿体转运肽与绿色荧光蛋白。这将证实预测的三个stc1等位基因的n端靶向序列是否在体内表现为叶绿体转运肽。拟议的活动将产生明显的更广泛的影响。本研究将遗传学、分子生物学和生物化学相结合，分析了玉米植物对害虫的反应。它将成为对应用生物化学和分子遗传学来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。私家侦探和合作私家侦探都是。是少数民族的成员，他们的实验室里充斥着代表性不足的群体。该项目与美国农业的长期改善有关，因为它解决了玉米保护自己免受昆虫攻击的机制。鉴定与昆虫抗性有关的基因将有助于育种者培育具有自然抗性的作物品种。了解主要作物的自然防御机制将有助于制定其他昆虫控制策略，并减少我们对潜在有毒农药的依赖。因此，要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.