【作者】 辛肇军；

【导师】 娄永根；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2011， 博士

【摘要】 植物的诱导抗性是植物抵御病虫害的一个重要机制,由于其具有广谱、持久的抗病虫特性,因此受到各国科学家的高度重视。建立筛选诱导植物抗性激发子的高通量模型,并在此基础上发掘激发子,对于阐述诱导抗性机理和改进病虫害治理具有重要意义。这一研究内容在植物与病原菌互作关系中已有较多的涉及,但在植物诱导抗虫性领域,至今尚没有这方面的研究报道。为此,本文选择芳樟醇合成酶基因(OsLIS),其催化产物为能引诱稻虱缨小蜂的芳樟醇,作为筛选诱导水稻抗虫性激发子的分子标记。通过融合OsLIS启动子区域与GUS报告基因,利用转基因技术获得转LISp：：GUS突变体,建立了高通量筛选诱导水稻抗虫性生物活性分子的模型。利用该模型,发现IAA的类似物2,4-二氯苯氧乙酸(2,4-Dicholorophenoxyacetic acid,2,4-D)可提高水稻抗虫性,并据此研究了其抗虫机制。同时,本文又克隆了水稻虫害特异性诱导表达的一个木聚糖酶抑制剂基因OsHI-XIP,发现过量表达该基因能增强水稻对害虫二化螟Chilo suppressalis (Walker)的抗性。因此,该基因可以作为今后筛选激发水稻诱导抗虫性活性分子的一个新型分子标记。本文的主要研究结果如下：根据OsLIS序列设计引物从水稻基因组中扩增了该基因转录起始位点上游1.5kb的启动子序列,命名为LISp。将LISp与GUS报告基因融合构建了LISp：：GUS表达载体,利用农杆菌介导的方法将表达载体转化水稻,获得了转基因品系LISp：：GUS,并利用LISp：：GUS品系建立了以根系吸收为测定方式,以GUS蛋白活性为检测指标的活性分子高通量筛选模型。经过大量筛选,发现了2,4-D可提高突变体中GUS蛋白活性。化学和转录分析表明外源施用2,4-D能提高水稻防御相关基因(OsMEK3, OsMPK3, OsMPK6, OsWRKY53, OsPLDa4)(?)信号途径基因(OsHI-LOX, OsACS2)的表达水平,能够增加茉莉酸(Jasmonic acid, JA)和乙烯(Ethylene, ET)的内源水平,并能诱导一些防御物质如胰蛋白酶抑制剂(Trypsin protease inhibitors, TrypPIs)和水稻挥发物的产生和释放,但是降低了二化螟取食诱导的水杨酸(Salicylic acid, SA)水平。与野生型水稻相比,在JA和ET合成途径阻断突变体(as-lox, as-pld (?)口as-acs)中,2,4-D诱导的OsLIS基因表达水平和TrypPIs水平显著下降。生物测定试验表明,外源施用2,4-D提高水稻对二化螟的抗性,并引诱褐飞虱Niaparvata lugens (Stal)的卵寄生蜂-稻虱缨小蜂Anagursnilaparvatae Pang et Wang;相反,外源2,4-D处理减弱了对褐飞虱的直接抗性。进一步的田间试验表明,在水稻上喷雾2,4-D可以引诱褐飞虱,并提高稻虱缨小峰对褐飞虱卵的寄生率,这一结果可以作为推-拉式策略防治褐飞虱的措施之上述结果表明,2,4-D可通过调节水稻的JA,SA和ET等信号途径对水稻的防御反应产生影响。克隆了水稻虫害特异诱导表达的一个木聚糖酶抑制剂基因OsHI-XIP,该基因cDNA全长1315bp,开放阅读框891bp,编码一个由297个氨基酸组成的蛋白质,预测分子量为32,5KDa。OsHI-XIP编码的蛋白定位于内质网,其转录水平在水稻根部要明显高于水稻的其它部位；二化螟取食、机械损伤和JA处理均能增加OsHI-XIP的表达水平。过量表达OsHI-XIP导致转基因水稻内源木聚糖酶抑制剂(Xylanase inhibitors, XIs),总活性上升,并增强水稻对二化螟幼虫的抗性,但不影响虫害诱导的水稻JA和TrypPIs含量。基于OsHI-XIP与水稻对二化螟抗性的相关性,OsHI-XIP可以作为今后筛选诱导水稻抗虫性激发子的一个新型分子标记。更多还原

【Abstract】 Induced resistance is an important mechanism of plant defense against disease and herbivorous insects. Because of its broad-spectrum and durability against pests, induced plant resistance was paid much attention by scientists. Exploiting synthetic chemical elicitors that trigger plant defense and new molecular markers that can be used to screen elictiors are of importance in elucidating mechanism of plant defense responses and in pest management. The work has been extensively carried out in the interaction between plants and pathogens. However, little to nothing was done in this regard in the field of plant induced defense against herbivores. In this study, we chosed a Linalool synthase gene (OsLIS), whose production linalool is a chemical that is attractive to the parasitoid, as molecular marker and established a high-throughput screening system, LISp::GUS homozygous rice plants with expression of the OsLIS promoter plus a reporter gene GUS, for synthetic chemical elicitors. Using this system, we identified one chemical,2,4-Dicholorophenoxyacetic acid (2,4-D) that can induce rice plant defense and thus we studied its elicitation mechanisms. We also cloned an herbivore specifically induced xylanase inhibitor gene OsHI-XIP. Overexpression of this gene increased rice resistance to the rice striped stem borer (SSB) Chilo suppressalis. This gene may be used as a new molecular marker for screening plant defense elicitors. The results are as follows:A 1.5kb promoter sequence upstream of OsLIS was amplified from rice genome based on the sequence of this gene and named it LISp. An expression vector LISp::GUS was conducted by fused LISp to GUS gene and transformed into rice by Agrobacterium-based transformation. Using transgenic lines, we established a specific high-throughput system for screening defense elicitors that trigger plant defense responses. One chemical,2,4-D, efficiently enhanced GUS activities in the transgenic lines. Chemical and transcriptional analysis show that exogenous application of 2,4-D up-regulated transcript levels of defense-related genes, such as three MAPK genes (OsMPK3,OsMPK6 and OsMEK3), OsWRKY53, OsHI-LOX, OsACS and OsNPR1, and enhanced levels of Jasmonic acid (JA) and ethylene (ET), and defense chemicals, Trypsin protease inhibitors (TrypPIs) and herbivore-induced volatiles but decreased Salicylic acid (SA) levels in herbivore-infested and non-infested plants. Moreover, the induction of 2,4-D on levels of OsLIS transcripts and TrypPIs decreased in mutants with impaired JA (as-lox and as-pld) and ethylene (as-acs) biosynthesis. Bioassay showed that exogenous application of 2,4-D increased rice plant direct resistance to the rice striped stem borer (SSB) Chilo suppressalis and indirect resistance to the rice brown planthopper (BPH) Niaparvata lugens, but decreased plant direct resistance to BPH. A field experiment found that there were higher population densities of BPH adults and higher parasitisms of BPH eggs by the parasitoid Anagrus nilaparvatae on rice plants sprayed with 2,4-D, which could be used to control BPH as one of "push-pull" means. These findings suggest that 2,4-D modulates rice defense responses by regulating the biosynthesis of JA, ET and SA.A 1315 bp cDNA of an herbivore specifically induced xylanase inhibitor gene OsHI-XIP, containing an open reading frame of 891bp which encodes a protein of 297 amino acids with a calculated molecular weight of 37.4KDa, was cloned. OsHI-XIP localized in endoplasmic reticulum (ER), and its expression levels in roots were higher than those in other organs. SSB feeding, BPH feeding, wounding and JA treatment up-regaulated expression levels of OsHI-XIP. Overexpression of OsHI-XIP enhanced rice resistance to SSB, but did not influence JA and TrypPI levels. These results suggested that OsHI-XIP was involved in rice plant defense against chewing herbivores and can be used as a new molecular marker for screening chemical elicitors.更多还原