摘要
芽孢杆菌(Bacillus spp.)是一种嗜热、好氧的革兰氏阳性细菌,该菌分布广泛,其能产生抗逆性强的芽孢、多种抗生素和酶类物质,是目前生防细菌中研究和应用较多的一类细菌。本研究首先通过实验室和温室试验,筛选并获得了对水稻细菌性病害有较好防治效果的芽孢杆菌生防菌株,同时通过生防菌株防治水稻细菌性病害的田间防治试验,综合评估了芽孢杆菌生防菌株的生防效果。为了解析芽孢杆菌与植物互作的分子机理,本研究利用蛋白双向电泳技术对芽孢杆菌处理后的水稻进行蛋白组水平上的差异比较分析,进而揭示芽孢杆菌对水稻的促生抗病机制。主要研究结果如下：
1.为了初步筛选用于防治水稻细菌性病害的芽孢杆菌生防菌株,我们在实验室条件下进行了平板抑菌实验和水稻促生实验。室内平板抑菌实验结果表明,实验室保存的14株芽孢杆菌菌株对水稻白叶枯病菌都有一定的抑菌效果,其中芽孢杆菌菌株90的抑菌效果最好,抑制率达到了60.87%。芽孢杆菌对水稻促生实验结果显示,芽孢杆菌处理对水稻有明显的促生作用,其中芽孢杆菌菌株FZB42对水稻的促生作用最为显著,水稻幼苗的株高和根长分别增加了40.9%和56.7%,芽孢杆菌菌株90、Gl-harpin、38和B3处理对水稻幼苗也有明显的促生效果,株高分别增加了31.5%、16.2%、10.0%和7.1%；根长分别增加了40.6%、26.4%、15.7%和19.1%。为了进一步评估室内试验筛选得到的芽孢杆菌菌株的生防效果,我们在温室条件下进行了芽孢杆菌防治水稻白叶枯病实验。从调查结果可知,芽孢杆菌菌株FZB42的防治效果最好,防效达到52.96%,芽孢杆菌菌株FZB24、90和38的防效也分别达到了41.01%、36.56%和30.33%。从2008年江苏省南京市和泗阳市的芽孢杆菌防治水稻白叶枯病田间试验调查结果可知,芽孢杆菌在田间自然条件下仍表现出较好的生防效果,芽孢杆菌处理的水稻叶部病情指数与对照处理相比均存在显著性差异；芽孢杆菌与化学农药交替使用能够显著提高水稻白叶枯病的田间防治效果。2009年江苏省泗阳市和湖南省益阳市的芽孢杆菌防治水稻细菌性条斑病田间结果显示,芽孢杆菌不仅对水稻细菌性条斑病也有较好的生防效果同时芽孢杆菌处理对水稻还有明显的增产作用。其中芽孢杆菌菌株B2和B9的田间防效分别达到了41.56%和36.71%,处理后的水稻产量分别增加了16.22%和14.39%。
2.目前,有许多研究报道了芽孢杆菌能够促进植物生长和诱导植物抗病性,但芽孢杆菌与植物互作机制还不是很清楚。本研究旨在利用蛋白双向电泳技术对芽孢杆菌处理后的水稻进行蛋白组水平上的差异比较分析,进而揭示芽孢杆菌对水稻的促生抗病机制。首先为了建立芽孢杆菌处理后水稻总蛋白高分辨率蛋白双向电泳体系,本研究对水稻总蛋白提取方法和蛋白双向电泳实验条件进行了优化。结果表明：采用优化后的TCA-丙酮沉淀法提取的蛋白样品质量更高,蛋白样品的2-DE图谱清晰,不同等电点和不同分子量的蛋白可以得到很好的呈现与分离。蛋白样品上样浓度为0.4mg/mL时,2-DE图谱分辨率高、蛋白点分布均匀。等电聚焦过程中低电压多步除盐和低电压聚焦的优化方法,可以有效减少等电聚焦过程所产生的横纹,获得更加清晰的2-DE图谱,实验结果表明适用于本研究的蛋白电泳聚焦程序为250V,1h;500V,1h;800V,1h;1000V,1h;8000V聚焦至60000Vh。
3.芽孢杆菌处理后,水稻根部和叶部中共发现了31个差异表达的蛋白点(22个上调表达,9个下调表达)。这些差异表达的蛋白包括了水稻的生长发育相关蛋白、抗病防卫反应相关蛋白、氨基酸代谢相关蛋白等。水稻细胞壁中的木葡聚糖转移酶在芽孢杆菌处理后明显的上调表达,在水稻生长发育过程中木葡聚糖转移酶参与了细胞壁葡聚糖代谢和细胞壁的调节过程,它的上调表达能够促进水稻叶部和茎部伸长从而促进水稻生长。芽孢杆菌处理后水稻中的植物抗病防卫反应相关蛋白如：谷胱甘肽-S-转移酶,过氧化物酶等也被诱导上调表达,这些蛋白的上调表达在蛋白组水平上解释了芽孢杆菌诱导水稻的抗病机制。
4.附录中介绍了研究生在读期间的另一部分工作,转harpinxooc蛋白编码基因大豆遗传转化体系的建立。这部分工作主要是对转harpinxooc蛋白编码基因大豆遗传转化体系和实验条件进行探索和优化,建立了两种以胚尖为外植体的大豆遗传转化体系,即：农杆菌介导以胚尖为外植体的大豆遗传转化体系和基因枪介导以胚尖为外植体的大豆遗传转化体系。同时本研究构建了植物高表达载体pCAMBIA3300::hrf2及农杆菌菌株EHA105(3300::hrf2);通过草丁膦抗性筛选获得了一定数量的大豆遗传转化幼苗；大豆转化幼苗的PCR检测结果显示,两种遗传体系均获得了表达harpinxooc蛋白的大豆转基因阳性植株。本研究为实验室后续的大豆遗传转化工作提供了很好的实验基础。
Bacillus spp. is a type of biocontrol bacteria, which is ideal for the biocontrol agent.since it has characteristics like promoting plant growth; inducing resistance; generating heat resistance, salt resistance, ultraviolet resistant endospore. The experiment aimed at screening Bacillus strains of our laboratory which have better control effect of rice bacterial diseases, through laboratory, greenhouse and field experiments.
1. It was showed with antagonistic activity on NA plates that, Bacillus strains preserved in our laboratory had good antibacterial effect of rice bacterial blight. Bacillus strain90showed antibacterial circle diameter28mm, the inhibition rate reached60.87%. Bacillus strain G1-harpin showed antibacterial circle diameter24mm, the inhibition rate reached52.17%. The inhibition rate of Bacillus strain38reached60.87%. The experiment of Bacillus on rice plant growth results showed that, shoot lengths and root lengths of rice seedlings were increased obviously after Bacillus treatment. Treatment with Bacillus strain FZB42resulted in greatest beneficial effects on rice growth, as root and shoot lengths increased by40.9%and56.7%, respectively. Bacillus strains90, G1-harpin,38and B3increased shoot lengths by31.5%,16.2%,10.0%and7.1%, respectively, while root lengths were increased by40.6%,26.4%,15.7%and19.1%, respectively. The experiment results of greenhouse and field control of rice bacterial diseases showed, Bacillus strains from our lab also had good control effect of rice bacterial disease under natural conditions, and the optimized formulation of Bacillus strains and chemical bactericide could significantly improve the field control effect.
2. Aimed to get the high-resolution two-dimensional gel electrophoresis system, the method of protein extraction and the experimental conditions were optimized in the present study. The results showed that the quality of rice protein using the optimized TCA-acetone precipitation method was better and the2-DE images were clearer, proteins with defferent isoelectric point and Molecular Weight were separated well. When the protein concentration loading for2-DE was0.4mg/mL, the good protein pattern with protein spots distributing equably and ayllabify could be obtained. The method of many steps wiping off salt and low voltage for isoelectro focusing in IEF process could reduce the cross striation and the good2-DE images were obtained.
3. Reports suggest that Bacillus spp. can be used to increase plant growth and resistance to disease, but the molecular mechanisms underlying the interaction between Bacillus spp. and plant is not completely understood. In the present study, to clarify these underlying mechanisms, the interaction between Bacillus spp. and rice was investigated using two-dimensional gel electrophoresis. Through comparative analysis, a total of31differentially expressed proteins were obtained upon Bacillus spp. treatment, including22proteins that were up-regulated and nine that were down-regulated. These data indicated that certain proteins involved in plant growth and development were up-regulated, such as xyloglucan endotransglycosylase. Interestingly, proteins involved in defense were also up-regulated, including peroxidases, glutathione S-transferases and kinases. Thus, proteins associated with disease resistance characteristics were induced in the plants after exposure to Bacillus spp. In addition, several proteins involved in protein-and lipid metabolism showed significant changes in expression. Overall, this present study demonstrates that Bacillus spp. can up-regulate the expression of proteins related to plant growth and defense, and lead to enhanced plant growth and disease resistance.
4. Appendix one showed one important part of my research work, tow soybean transformation systems for expression of harpinxooc protein were established. This study aimed to optimize the experimental conditions in the soybean transformation process, establish tow soybean transformation system used soybean embryonic tip, agrobacterium-mediated soybean transformation and biolistic-mediated soybean transformation. The vector pCAMBIA3300::hrf2and agrobacterium strain EHA105(3300::hrf2) were designed for soybean transformation. Following the glufosinate selection, many acclimatized plantlets were obtained. The results of PCR detection for soybean transgenic seedlings showed that transgenic positive plants were obtained using these two soybean transformation systems. The research established the experimental foundation for next work.
引文
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