【作者】 毛碧增；

【导师】 何祖华； 李德葆；

【作者基本信息】 浙江大学 ， 植物病理学， 2008， 博士

【摘要】 水稻纹枯病（Rhizoctonia solani）、稻瘟病（Magnaporthe grisea）和白叶枯病（Xanthomonas oryzae pv.oryze）是我国水稻的“三大”病害。由于水稻种质库中尚未发现有对纹枯病可利用的高水平抗源、稻瘟病生理小种变异快,这使得选育具有广谱抗性并且可以稳定遗传的抗病品种显得异常重要。利用外源几丁质酶基因和葡聚糖酶基因提高水稻抗性是一种可能的选择。本实验室的前期工作中,首次将水稻几丁质酶基因（RCH10）和苜蓿β-1,3-葡聚糖酶基因（AGLU1）双价基因表达盒（Z100）转化水稻,经初步温室检测提高了对水稻纹枯病和稻瘟病的抗性。超表达的OsNPR1转基因植株显著增强了对白叶枯病的抗性,但转基因植株有矮化,结实率降低现象。本研究对Z100转基因水稻后代进行进一步的田间抗病以及表达盒中基因的遗传稳定性进行分析。经PCR和GUS检测,结果表明T₁代转基因植株存在分离现象,分离比例具有多样性,有的符合孟德尔3:1、15:1,有的不符合遗传规律,揭示了外源基因在基因组染色体上插入位点的随意性;在T₃代转基因株系还发现NPT||基因100%传递给子代,而整个表达盒传递的为80%,其中RCH10基因丢失了15%,AGLU1基因丢失了5%,说明T-DNA上的遗传信息在子代发生了丢失。经过4代的纹枯病和稻瘟病抗性检测,获得5个双抗株系,经Southern检测,表明外源基因已整合到基因组中,Northern杂交结果表明两个基因表达水平相同且与抗病能力存在剂量效应。同时发现纹枯病和稻瘟病接种诱导了OsPR1b基因的表达,转基因植株中表达水平高于野生型,说明转基因加强了水稻自身防卫基因的表达,并可能与转基因一起具有协同抗病功能。利用免疫金标记技术,跟踪了病原菌接种后RCH10和AGLU1在转基因植株中的的亚细胞定位和动态表达,结果发现转基因植株中金颗粒在叶绿体内的分布密度远远高于其他细胞器;Z100转基因植株在受到病原菌侵染24 h后,抗菌蛋白会从叶绿体向细胞质和细胞壁分泌,最终可能聚集在病原菌侵染部位,行使杀菌功能,初步明确了抗病转基因植株中外源蛋白表达、积累以及转运的时空特点、表达量与抗病性的关系,这一现象尚属首次发现。转基因在Z100转基因植株中酶活力显著高于野生型,但在诱导后的不同时间段表达一直较高且水平相近,这是因为受组成型启动子或增强子调控所致。氧化酶系统中,Z100抗病转基因水稻在纹枯病侵染后,丙二醛（MDA）含量增加和SOD的活性增强,揭示了转基因植物消除活性氧能力可能得到加强,减轻了对细胞膜的伤害,从而增强了抗病性。Z100转基因对植株千粒重和苗期发育等农艺性状有影响。转基因植株种子的千粒重比野生型显著减小,发芽势和发芽率也比野生型低,尤其对GA₃处理敏感。基因表达分析结果表明GA信号途径基因GID1、SLR1、D1和GID2的转录水平在转基因植株和野生型中相似,而GA2ox1在转基因植株中的转录水平显著高于野生型。因此Z100转基因可能促进了苗期GA的失活,转基因植株早期苗期的植株矮小,可能与GA2ox1基因的高水平表达促使GA降解,导致内源活性GA缺乏有关。NPR1是植物系统获得性抗性（SAR）的关键调控因子。本实验室前期的工作证明了水稻ortholog OsNPR1的功能,发现OsNPR1转基因水稻对白叶枯病具有高度抗性,水稻中可能存在由水杨酸（SA）介导的SAR途径。但后续的研究发现高抗的转基因植株存在与Z100转基因类似的植株变矮性状,即OsNPR1转基因也影响了其它农艺性状的发育。为了进一步了解OsNPR1诱导的防卫反应和发育调节,对过表达OsNPR1基因（OsNPR1-OX）的矮化转基因水稻采用全基因组水稻基因芯片进行分析,发现OsNPR1抗病转基因引起差异表达基因共968条,其中上调796条,下调172条,其中部分基因与发育有关。初步揭示了OsNPR1调节的植物生理学过程及一些关键的目标基因。本研究的结果对于研究RCH10和AGLU1双价基因共转化和OsNPR1在抗性以及发育上的调控机制提供了新的方向,同时也提供了具有较高应用价值的高抗转基因株系,具有一定的育种应用价值。对于进一步从分子生物学上阐明抗病性激发对其它农艺性状的影响,为更好地进行抗病分子设计奠定了基础。更多还原

【Abstract】 The rice sheath blight （sb） caused by Rhizoctonia solani Kuhn, rice blast caused by Magnaporthe grisea and bacterial blight （bb） disease caused by Xanthomonas oryzaepv oryzae （Xoo） are the most destructive diseases in rice. In particular, there is no genetic resource resistant to sheath blight, and the complex genetic diversity of M. oryzae, genetic transformation with chitinase and glucanase genes to enhance resistance to those fungal provides an alternative approach. In the previous work of our laboratory, the rice chitinase gene （RCH10） and the alfalfaβ-1,3-glucanase gene （AGLU1） were tandem-inserted into the transformation vector pBI101 under the control of the 35S promoter with its enhancer sequence to generate double-defense gene expression cassette pZ100. The pZ100 cassette was transformed into rice （cv. Taipei 309） by Agrobacterium-mediated transformation, transgenic plants enhanced resistance to both sheath blight under greenhouse condition. Over-expression of OsNPR1 gene （OsNPR1-OX）, a key regulator in plant systemic acquired resistance （SAR）, transgenic plants increased resistance to bacterial blight. Both Z100 and OsNPR1-OX plants appeared stunt and seed setting reduction.In this study, we analysed genetic stability of Z100 transgene, and possible mechanism of development modification in Z100 and OsNPR1-OX plants.More than 160 Z100 independent transformants were obtained and confirmed by PCR and GUS analysis. The segregation ratio of some T₁ transgenic rice plants showed 3:1 and 15:1 with Mendelian inheritance pattern, but other lines didn’t, indicating that the transgene inserted into rice chromosomes with a complex and casualness. Wefurther observed 100% the NPTII marker gene in the offspring （T₃）, but RCH10 genewas lost at 15% cases and AGLU1 gene at 5% cases during progeny generating. Northern blotting analysis of inheritable progenies revealed similar levels of the RCH10 and AGLU1 transcripts in the same individuals. Disease resistance to both sheath blight and blast was enhanced in open field inoculation an evaluation valuable to rice breeding. OsPR1-b was stronger induced in the Z100 transgenic plants.Immunogold detection revealed that RCH10 and AGLU1 were located mainly in the chloroplasts in the transgenic plant, and they were delivered to the vacuole and cell wall after 24 h after infection by R. solani, suggesting that these antifungal proteins might gather and execute action in these subcellular compartments. The results probably suggest a novel mechanism for defense protein function during pathogens attack, we propose that following pathogen invasion, defense protein maybe trigged and moved to the attacked sites to play antifungal function.The expression and activity of chitinase and glucanase were investigated in transgenic plant resistant to sheath blight and the wild-type （WT） inoculated with R. solani, the results indicated that two enzymes in transgenic plant have samilar transcription levels among infection phase, and activities were significantly higher than that of WT. Membrane lipid peroxidation and antioxidant enzymes were measured in transgenic rice and the wild-type （WT） inoculated with R. solani. The results indicated that malondialdehyde （MDA） generation was induced by pathogen infection. Compared with the WT, the MDA content in the transgenic plants was significantly lower with or without pathogen inoculation. Superoxide dismutase （SOD） activity was significantly stimulated by pathogen in transgenic rice compared to the WT. The results suggest that MDA content and SOD activity might play roles in resistance enhanced by the transgene.We also observed that transgenic seeds displayed lower germination ratio and seedling vigor, and seedlings were more sensitive to exogenous GA application compared with the WT. Gene expression detected by RT-PCR showed that GID1, SLR1, D1 and GID2, the key GA signaling genes had the expression levels same to the WT, but the GA catabolism GA2oxl gene transcription level was significantly higher in the Z100 plants than that in WT plants. These results indicated that plant growth and development stunt might attributed to deactivation of bioactive GA by the transgenes, GA2oxl expression would decrease the level of endogenous bioactive GA.In order to get a fundamental sight into the OsNPR1-mediated development abnormality, a rice whole-genome chip representing 51 279 units was used to profile globle gene expression patterns in OsNPR1-OX transgenic plants, we obtained 968 differentially expressed in OsNPR1-OX genes versus WT, the statistical data indicated that 796 genes exhibited up-regulated and 172 genes were down-regulated, and some genes were involved in the plant development.These studies provide molecular clues of the defense transgene-mediated development modification, and target genes for the further studies.更多还原