【作者】 王劲波；

【导师】 朱昌雄； 王慧敏；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2002， 硕士

【摘要】 中生菌素是一种非常有应用前景的农用抗生素类生物农药，但是对中生菌素的作用机制我们还没有深入的了解。长期大规模的田间应用表明农用抗生素在田间防治有些病害时具有比化学农药更好的防治效果，而离体抑菌（杀菌）效果却不及化学农药；预防效果好于治疗效果；研究还发现长期应用这些农用抗生素，病原菌也未出现抗药性。所有这些研究结果和现象都说明农用抗生素不仅直接抑制或杀死病原菌，而且可能对植物体也发生了作用，提高了植物体的抗病性。因此我们试图在水稻抗、感白叶枯病菌近等位基因系悬浮细胞体系中，用RNA斑点杂交方法研究中生菌素对重要的植物防卫反应基因过氧化物酶（PO）基因、苯丙氨酸解氨酶（PAL）基因、查尔酮合成酶（CHS）基因转录表达的影响，从而阐明中生菌素的作用机制。 首先，我们对粳稻品种感病品系沈农1033及其携带Xα-4抗病基因的近等位基因系CBB₄的愈伤组织培养、悬浮细胞系的建立进行了探索。结果表明：水稻品种的遗传特性、2,4-D浓度是诱导水稻愈伤组织、水稻悬浮细胞系培养的关键因素。根据以上研究基础，建立了一套感病品种沈农1033和抗病品种CBB₄的愈伤组织培养、悬浮细胞系培养的程序，对这一程序前人未有完全一致的报道。 其次，我们在水稻抗、感白叶枯病菌近等位基因系悬浮细胞体系中，研究白叶枯病菌在水稻细胞悬浮液中的生长情况及中生菌素对PO基因、PAL基因、CHS基因诱导转录表达的影响，结果表明： 在接种白叶枯病菌后，水稻感病品种悬浮细胞系中的白叶枯病菌的数量远多于抗病品种悬浮细胞系，这说明水稻抗病品种悬浮细胞系与感病品种悬浮细胞系相比有明显的抑制细菌的生长和繁殖的作用，即水稻对白叶枯病抗性与抑菌作用有关。 在接种白叶枯病菌后，水稻抗病品种PO基因、PAL基因、CHS基因这3个重要的植物防卫反应基因诱导转录表达比感病品种早，而且表达的量大，这说明PO基因、PAL基因、CHS基因诱导转录表达量与水稻品种的抗病性呈正 中国农科院研究生院 沏＊：学位论义相关，水稻PO基因、PAL基因、CHS基因诱导转录表达量也许能作为水稻白叶枯病抗性鉴定的指标。 无论白叶枯病菌存在与否，中生菌素对感病品种沈农1033和抗病品种CBB。的PO基因、PAL基因的转录表达均有较强的诱导作用；但中生菌素对抗病品种CBB。的CHS基因的转录表达的诱导作用比对感病品种沈农1033诱导作用弱。这说明中生菌素即能抑制水稻白叶枯病菌的生长，又能显著地诱导PO基因、PA L基因、＊＊8基因这3个重要的植物防卫反应基因的转录表达，而且对抗、感白叶枯病菌水稻品种有相近的诱导效果。PO基因、PAL基因、CHS基因参与的植物防卫反应中的木质素前体物质、酚类物质、自由基、活性氧、植保素等物质的合成，这些物质都具有抗菌活性。因此，中生菌素诱导和增强了植物的防卫反应，增强了植物的抗病性。 通过对中生菌素处理的水稻悬浮细胞体系中植物防卫反应基因转录表达规律的研究，不仅为中生菌素的作用机制的阐明奠定了基础，同时也为建立新型农用抗生素杀菌剂筛选模型，为我国丰富的微生物资源的开发利用提供新的技术手段；也为中生菌素适时、适量及适当的田间应用技术提供理论指导。更多还原

【Abstract】 Zhongshengmycin (a new, commercially available agricultural antibiotic from 1996) has been widely utilized for controlling plant diseases, particularly on bacterial diseases. However the mode of action of Zhongshengmycin do not completely be understood. The previous research had discovered that Zhongshengmycin was less to inhibit the growth and reproduction of Xanthomonas oryzae pv. oryzae (Xoo) than chemical pesticides, but Zhongshengmycin protected Xoo infection to rice better than chemical pesticides. In addition, the resistance of plant pathogens to Zhongshengmycin is not found up to now. In order to settle the query, temporal accumulation of several important defense gene transcripts in suspension-cultured cells of the near-isogenic lines for bacterial blight resistance of rice was detected.The results showed that genetic trait of rice and quantity of 2,4-D were the key elements to callus induction from mature rice seeds and suspension culture of rice cells. The procedure of callus induction from mature rice seeds and suspension culture of rice cells was set up.Kinetics of Xoo multiplication in rice suspension cells was studied. The quantity of Xoo multiplication was obviously less in the rice suspension cells of resistant rice cultivar CBB4 than in the rice suspension cells of susceptible rice cultivar Shennong 1033. The result indicated that inhibition of Xoo multiplication in the rice suspension cells was related to rice resistance to Xoo.The transcripts of PO, PAL and CHS genes, three defense genes encoding respectively peroxidase (PO), phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) in suspension-cultured cells of the near-isogenic lines for bacterial blight resistance of rice were discovered during interactions with Xoo. Northern blot analysis showed that the accumulation of PO, PAL and CHS gene transcripts in susceptible rice cultivar Shennong 1033 was generally delayed, attenuated and lasted for a shorter period of time as compared with the accumulation of PO, PAL and CHS gene transcripts in resistant rice cultivar CBB4. The accumulation of PO, PAL and CHS gene transcripts in suspension-cultured cells of the near-isogenic lines for bacterial blight resistance of rice was positively related to rice resistance to bacterial blight disease, and was analyzed as the indicator of rice resistance to bacterial blight disease.The transcripts of PO, PAL and CHS genes in suspension-cultured cells of the near-isogenic lines for bacterial blight resistance of rice were detected during interactions with Zhongshengmycin. Northern blot analysis suggested that theABSTRACTbactericide Zhongshengmycin could greatly strengthen PO, PAL gene transcripts in the rice suspension cells of susceptible rice cultivar Shennong 1033 and resistant rice cultivar €884 regardless of the presence or absence of Xoo. During interactions with Zhongshengmycin, the accumulation of PO and PAL gene transcripts in the rice suspension cells of susceptible rice cultivar Shennong 1033 and resistant rice cultivar €884 was markedly a higher level than interactions between rice and Xoo. In addition, the bactericide Zhongshengmycin could greatly strengthen CHS gene transcripts in the rice suspension cells of susceptible rice cultivar Shennong 1033 regardless of the presence or absence of Xoo, while the transcripts of CHS gene in the rice suspension cells of resistant rice cultivar CBB4 accumulated moderately in response to Zhongshengmycin regardless of the presence or absence of Xoo. Interestingly, during interactions with Zhongshengmycin all these transcripts of PO, PAL and CHS genes in suspension-cultured cells of the near-isogenic lines for bacterial blight resistance of rice accumulated to a higher level than the interactions between rice and Xoo regardless of the presence or absence of Xoo.The findings of this study demonstrated that Zhongshengmycin could not only inhibit the growth and reproduction of Xoo, but also induce the accumulation of PO, PAL and CHS gene transcripts in suspension-cultured cells of th更多还原