水稻抗病相关基因的分离克隆和功能鉴定_水稻已克隆基因表

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论文中英文摘要

作者姓名：丁新华

论文题目：水稻抗病相关基因的分离克隆和功能鉴定

作者简介：丁新华，男，1980年06月出生，2002年09月师从于华中农业大学王石平教授，于2008年06月获博士学位。

中

文

摘要

水稻白叶枯病和稻瘟病分别是由白叶枯病菌（Xanthomonas oryzae pv.oryzae, Xoo）和稻瘟病菌（Magnaporthe grisea）引起，是世界水稻生产中的两大重要病害，造成巨大的损失。通过改良水稻自身防御体系来有效的控制病害，是一种即经济又“绿色”的方法。鉴定水稻抗病相关基因，研究水稻抗病机理对改良水稻有着重要的科学意义和应用前景。

植物激素生长素诱导的信号通常被认为调节植物的生长和发育。本研究报道的水稻基因GH3-8是一个生长素反应基因，在依赖于生长素的发育中发挥功能，同时也调节不依赖于水杨酸和茉莉酸的信号路径的抗病反应。白叶枯病病菌诱导水稻至少在被侵染部位合成生长素，而生长素继而诱导水稻大量合成松弛细胞壁的蛋白质－伸展蛋白（α-和β-expansins），破坏细胞壁对病原菌的先天屏障作用，以利病原菌在水稻中生长繁殖。在携带有抗病基因Xa21或Xa26抗病水稻品种中，病原菌引起的水稻感染部位生长素的合成可诱导水稻快速合成IAA酰胺合成酶GH3-8。GH3-8通过催化IAA－氨基酸的合成抑制生长素的作用，从而阻止细胞壁的松弛，增强植物对病原菌的自身免疫功能。超量表达GH3-8基因增强水稻对白叶枯菌的抗性，同时也延缓了植株的生长和发育，至少部分因为抑制了生长素信号从而抑制了α-和β-expansins。本研究结果揭示了病原菌利用生长素作为毒性因子侵染水稻的机理、以及水稻应对这一毒性因子的调控途径，同时也从一个方面解释了植物在抗病反应中通常要付出生长被抑制的代价的原因。

超量表达GH3-8导致植株不育。通过正反交试验显示GH3-8超量表达植株是雄性和雌性都不育。通过形态学观察发现GH3-8超量表达植株的雌蕊柱头发育不正常；通过激光共聚焦显微镜对雌蕊胚囊观察发现，GH3-8超量表达植株的成熟胚囊发育不正常，这可能是GH3-8超量表达植株雌性不育的原因。通过形态学观察发现GH3-8超量表达植株的雄蕊和野生型无异，但花粉碘染显示GH3-8超量表达植株大部分花粉都败育，这可能是GH3-8超量表达植株雄性不育的原因。通过分析GH3-8基因表达模式显示GH3-8基因特异在雄蕊高表达，并随着花的发育表达强弱也不断变化，而在雌蕊基本检测不到表达。组织和时间的特异表达也印证了GH3-8在调控花的发育中作用。利用酵母单杂交技术，筛选得到和GH3-8基因启动子互作的几个生长素反应因子。其中OsARF8超量表达激活GH3-8基因的表达，证明OsARF8是调控GH3-8基因表达的转录因子。通过分析OsARF8基因表达模式显示OsARF8基因特异在雌蕊高表达，而在雄蕊表达很低。OsARF8基因超量表达植株和野生型植株相比育性下降。花粉碘染显示OsARF8基因超量表达植株大部分花粉败育；检测雌蕊没有发现和野生型有显著

差异。花粉的育性下降可能是OsARF8超量表达植株育性下降的原因。生长素信号路径中的基因（OsARF8和GH3-8）的不正常表达影响了水稻育性，说明生长素信号可能在调控水稻育性中有重要作用。检测水稻穗发育中生长素分布也显示生长素和穗的发育密切相关。

在水稻品种明恢63中抑制一个维生素B1合成基因OsDR8的表达，显著提高了转基因植株对白叶枯病和稻瘟病的敏感。外源应用维生素B1可以互补抑制OsDR8基因对水稻植株抗病的影响。几个防御反应基因包括防御信号路径的早期功能基因OsPOX和OsPAL基因以及路径下游基因OsPR1a、OsPR1b、OsPR4、OsPR5 和OsPR10的表达在OsDR8抑制表达的植株中下降。这些结果说明OsDR8影响植株的抗性可能是通过影响防御反应基因的表达，OsDR8的功能在信号路径的上游。另外，维生素B1的积累可能是水稻植株对白叶枯病和稻瘟病的抗性所必须的。

通过筛选水稻T-DNA插入突变体库，发现一个类病斑突变体。侧翼序列分析显示T-DNA插在一个基因（命名为OsDR9）的开放读码框。预测的OsDR9基因编码由180个氨基酸组成的功能未知蛋白。OsDR9基因在茎和幼穗中表达很低，而在幼苗、剑叶、叶鞘和愈伤表达较高，在根中没有检测到表达。另外OsDR9基因在老叶中比新叶表达更高。突变体对稻瘟病和胡麻叶斑病表现高抗。对有类病斑的叶片进行组织化学检测和DNA断裂分析显示细胞死亡具有凋亡特征。病程相关蛋白PR4和PR8以及稻瘟病相关基因AOS2在突变体中上升表达。突变体植株也积累了自发荧光物质、SA、JA和植保素（momilactone A 和 sakuranetin）。突变体提高了超氧化物和H2O2的水平。将一个来源于水稻品种日本晴的包含有OsDR9基因的10.5kb片段转入突变体02Z15AM37，转基因植株类病斑表型消失，说明由于T-DNA插入导致的OsDR9突变是是引起类病斑表型的原因。这些结果说明OsDR9是水稻抗病和细胞凋亡的一个负调节子。

关键词：

水稻；白叶枯病；稻瘟病；抗病相关基因；生长素；水杨酸；茉莉酸；基础抗性；发育；伸展蛋白；GH3；维生素B1；类病斑突变体；凋亡；植保素；活性氧物质

Isolation and Functional Characterization of Pathogen-induced Defense-responsive Genes of Rice

Xinhua Ding

ABSTRACT Rice bacterial blight disease caused by Xanthomonas oryza sative pv.oryza and fungal blast disease caused by Magnaporthe grisea, are two of the most devastating diseases of rice worldwide.These two diseases cause tremendous yield lo each year.Efficient control of disease through improving rice defensive system is economic and green way.Characterizing rice disease resistance related genes and elucidating the mechanism of rice disease have important significance in science and application for improving rice.The signaling induced by the plant growth hormone auxin is generally recognized to regulate plant growth and development.Here we report that rice GH3-8, an auxin-responsive gene functioning in auxin-dependent development, activates disease resistance in a salicylic acid– and jasmonic acid–signaling-independent pathway.Bacteria induce accumulation of indole-3-acetic acid(IAA), the major type of auxin, in rice.IAA induces the expreion of α-and β-expansins, the proteins that are known to loose cell wall, the native barrier of biotic intruder, to facilitate the growth of cells.In resistance rice variety carrying Xa21 or Xa26, the infection of bacteria induce rice to synthesize GH3-8 in infection site of rice.GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation and loosene of cell wall.Overexpreion of GH3-8 results in enhanced disease resistance and retarded growth and development, which is at least partly due to inhibiting the expreion of α-and β-expansins via suppreing auxin signaling.Here we show the mechanism of bacteria hijacks auxin as virulence factor to infect rice, and the regulating pathway of rice to the virulence factor;in addition to, explain the cause that plants growth was restrained in disease resistance.Overexpreion of GH3-8 results in sterility of plants.Analysis of forward cro and reverse cro showed that GH3-8-overexpreing plants were male sterility and female sterility.We found that the stigmas of GH3-8-overexpreing plants are abnormal by morphological observation.We observed the mature embryo sac of GH3-8-overexpreing plants using laser scanning confocal microscopy.The result showed that the mature embryo sacs of GH3-8-overexpreing plants were abnormal.This may be the reason of female sterility.No obvious difference was observed in stamen between GH3-8-overexpreing plants and wide type in stamen, but the most of pollen of GH3-8-overexpreing plants were sterile.This may be the reason of male sterility.GH3-8 had high expreion level in stamen, and the expreion of GH3-8 changes as the development of flower.Tiue and time differential expreion confirmed the role of GH3-8 in regulating flower development.We gained several auxin responsive factors(ARFs)that interacted with the promoter of GH3-8 by analysis of yeast one hybrid.Overexpreion of OsARF8 in Mudanjiang 8 activated the expreion of GH3-8.This result suggested that OsARF8 is the transcription factor in regulating the expreion of GH3-8.OsARF8 had high expreion level in pistil, and very low expreion level in stamen.GH3-8-overexpreing plants had lower fertility than wide type.The most of pollen of OsARF8-overexpreing plants were sterile.The overexpreion of Auxin signaling genes(OsARF8

and GH3-8)resulted in decrease of rice fertility.This result suggested that auxin plays a critical role in regulating flower development.The detection of the auxin distribution in panicle development showed that auxin is affinitive relation with panicle development.The transgenic plants with repreed expreion of OsDR8 showed reduced resistance or susceptibility to Xanthomonas oryzae pv.oryzae and Magnaporthe grisea causing bacterial blight and blast, respectively.The putative product of OsDR8 was highly homologous to an enzyme involved in the biosynthesis of the thiazole precursor of thiamine.Exogenous application of thiamine could complement the compromised defense of the OsDR8-silenced plants.The expreion level of several defense-responsive genes including the earlier functional genes of defense transduction pathway, OsPOX and OsPAL, and the downstream genes of the pathway, OsPR1a, OsPR1b, OsPR4, OsPR5 and OsPR10, was also decreased in the OsDR8-silenced plants.These results suggest that the impact of OsDR8 on disease resistance in rice may be through the regulation of expreion of other defense-responsive genes and the site of OsDR8 function is on the upstream of the signal transduction pathway.In addition, the accumulation of thiamine may be eential for bacterial blight resistance and blast resistance.We found a mutant with lesion mimics on the leaves through selecting a T-DNA insertional rice pool.The inserted T-DNA was inserted into the open reading frame(ORF)of a gene named OsDR9.The predicted encoding product of OsDR9 consists of 180 amino acids that is an unknown functional protein.OsDR9 had very low expreion level in the stem and young panicle but higher level in seedling, flag leaf, sheath and callus;no OsDR9 expreion was detected in the root.In addition, OsDR9 had higher expreion level in old leaf than young leaf.The mutant was highly resistance to Magnaporthe grisea causing fungal blast disease and bipolaris oryzae causing Cochliobolus miyabeanus disease in field.Histochemical detection and DNA fragmentation of the leaves developed lesion mimics showed that the cell death had the same features of apoptosis.In addition, the expreion of pathogenesis related(PR)proteins genes PR4 and PR8 as well as a blast resistance related gene AOS2 was upregulated in the mutant.The mutant also accumulated autofluorescent materials, salicylic acid and phytoalexins(both momilactone A and sakuranetin).The mutant contained elevated levels of superoxide and H2O2.A 10.5-kb fragment harboring the OsDR9 gene from rice variety Nipponbare was transferred into the mutant.Lesion mimic phenotype was disappeared in the transgenic plants, indicating that knockout of OsDR9 by T-DNA insertion caused the lesion mimic mutant phenotype.These results suggest that OsDR9 is a negative regulator in rice disease resistance and apoptosis.Key words: Oryza sativa;bacterial blight;fungal blast;auxin;salicylic acid(SA);jasmonic acid(JA);basal resistance;development;expansin;GH3;thiamine;lesion mimic mutant(LMM);apoptosis;phytoalexin;reactive oxygen species(ROS)