【作者】 陈晓婷；

【导师】 王宗华；

【作者基本信息】 福建农林大学 ， 农药学， 2008， 博士

【摘要】 许多在生态、生理学上不同的真菌都可以产生草酸（Oxalic acid, OA）。这些草酸产生菌如核盘菌、灰葡萄孢等寄主范围广泛,可以引致上百种植物病害,造成世界范围的粮油、蔬菜等农作物的严重减产。草酸在病菌致病过程中可能起着关键性的作用。因此研究植物对草酸产生菌的防卫机制,挖掘抗病资源,提高农作物抗性,是急需解决的重要科学问题,也是生产上首先要解决的实践问题。拟南芥具有基因组小、结构简单、形体小、生长周期短、繁殖系数高、自花授粉、基因组序列已知、与高等植物的基因组间有较高同源性等特点,是研究植物防御机制的良好模式植物。因此本论文着眼于拟南芥与草酸的互作机制的研究。分为三部分:1.通过筛选草酸抗性突变体,并对突变体进行分子分析。最终目的是分离草酸抗性基因来获得作物的菌核病抗性,从而减少农药的使用,提高环境的质量和食品安全;2.草酸可以通过氧化与脱羧两条途径降解,本研究从枯草芽孢杆菌中克隆草酸脱羧酶,从大麦中克隆草酸氧化酶,构建其植物表达载体,转化拟南芥,对转基因植株进行菌核病抗性分析;3.对草酸胁迫下拟南芥全基因表达谱和microRNA的差异表达谱进行分析,进一步明了草酸致病的分子机制,为草酸产生菌的生物防治提供理论基础。获得的主要结果如下:1.通过拟南芥Col-0的浓度梯度的实验,确定了筛选草酸抗性突变体的筛选压为1.2mmol/L OA;建立并优化了草酸抗性突变体的筛选体系;2.通过对中国科学院遗传与发育生物学研究所左健儒实验室构建的化学诱导型功能获得和缺失性突变体库（公开释放的6,000余个突变体）进行两代的筛选,得到5株草酸抗性增强突变体,并通过TAIL-PCR扩增获得T-DNA插入的侧翼序列;BLAST结果显示这5株突变体中有4株突变体（D630、D282、D154、D74）均插在At5g10450的第一个内含子上,并且验证了At5g10450基因的SALK T-DNA插入缺失突变体的SALK₀68774（插入位点为外显子）的纯合体并没有草酸抗性。D33突变体插入位点为At2g39720和At2g39730两个基因间区;3.半定量RT-PCR表明D33突变体的At2g39690、At2g39700、At2g39720及At2g39750表达上调;4.构建了At2g39690,At2g39700和At2g39720的过表达载体,进行了拟南芥浸花转化,将对转基因植株进行菌核病抗性和草酸抗性分析;5.对突变体分别进行了核盘菌的离体叶片接种和活体接种,发现无论是离体接种还是活体接种,突变体都比野生型植株更抗菌核病。说明通过筛选草酸抗性突变体来筛选抗菌核的材料是可行的。有可能通过分离草酸抗性基因来获得作物的菌核病抗性,从而减少农药的使用,提高环境的质量和食品安全;6.从枯草芽孢杆菌Bs168中克隆了草酸脱羧酶基因（Yvrk）,从大麦中克隆了草酸氧化酶基因（Y14203）并构建了植物表达载体,对拟南芥进行转化,获得了转基因植株。转草酸脱羧酶的拟南芥植株表现出对核盘菌的抗性,这为草酸产生菌的生物防治提供了一条新途径;7.首次利用拟南芥全基因组Oligo芯片研究草酸胁迫下差异基因表达谱。通过对两张生物学重复的表达谱芯片数据的生物信息学分析表明:草酸胁迫诱导了拟南芥的JA/ET途径,而不是SA途径。此外,苯丙烷代谢途径、WRKY转录因子家族、细胞色素P450家族、ABC转运蛋白、热激蛋白基因家族上调表达,表明这些途径或基因家族参与了拟南芥对草酸的抗性反应。8.首次利用拟南芥microRNA芯片研究草酸胁迫下microRNA差异基因表达谱。获得三个差异表达microRNA和9个差异表达的Predicted_miRNA。其中下调表达的小立碗藓Ppt-miR1211,根据miRU找到一个最匹配的靶mRNA是一个萜烯合成酶（At3g14520）;另一下调表达的毛果杨Ptc-miR3991 （与Ath-miR399b同源）,对应的靶mRNA是一个泛素连接酶（At2g33770）;这两个靶基因在表达谱芯片中的Ratio值都上调。上调表达的Ath-miR858有三个靶mRNA,分别为At2g47460（转录因子）、At3g08500（转录因子）、At3g20310（ethylene responsive element- binding family protein）,它们在表达谱芯片中对应的Ratio值都分别下调。说明microRNA对其靶mRNA确有负调控功能。更多还原

【Abstract】 Many physiologically and ecologically different fungi can secrete oxalic acid (OA), which plays a crucial role during pathogen infection process. OA-producing pathogens, such as S. sclerotiorum and Botrytis cinerea, can infect hundreds of plants and cause severe loss of crops, but the material resistant to the fungi is limited due to the extensive host range. So, there is a great need to study the defense response to OA-producing pathogen and seek the resistant materials. Arabidopsis thaliana possesses good characteristics, such as small genome, simple structure, small figure, short life period, high propagating rate and self-pollination etc. Moreover, its genomic sequence was completed on the end of 2000, which provided high homology between Arabidopsis and higher plants on the genome. So, Arabidopsis has been used as a model plant for the analysis of plant defense response. In this paper, the interaction between Arabidopsis and OA was studied in three aspects: first, OA-resistant mutants were obtained from screening a chemical-inducible mutant library and their molecular mechanism was studied. The ultimate objective is to isolate the OA-insensitive gene and obtain the S. sclerotiorum resistant material, thus, decrease the use of pesticide and improve the quality of environment and security of foods; second, Oxalic acid is catabolized by two major pathways, i.e. decarboxylation and oxidation. So, oxalate decarboxylase was cloned from Bacillus subtilis 168, and oxalate oxidase was cloned from barley, and their plant expression vectors were constructed and transgenic plants were obtained by floral dip. The S. sclerotiorum resistance was detected among the transgenic plants; third, oligo microarray and microRNA microarray were used to investigate the differentially expressed genes and miRNAs under the stress of OA, thus further understand the mechanism of pathological effects of OA and provide the theoretical foundation of biological control on OA-producing pathogens.The major results in this study are as follows:1. Based on the effects of different concentration OA treatments on the growth of Arabidopsis ecotype Columbia-0 seeds, the threshold concentration of OA was obtained. The screen system was to sow the sterilized seeds of mutant library on agar plates of MS medium (pH 6.5, calcium free) supplemented with OA (final concentration 1.2 mmol/L), estradiol (final concentration 10μmol/L), and MES (final concentration 1g/L).2. Putative OA-insensitive mutants were screened from the mutant library constructed by the laboratory of Professor Zuo Jiruo from the Chinese Academy of Sciences (about 6000 individual lines) using MS medium containing 1.2 mmol/L OA and 10μmol/L estradiol. About 300 putative OA insensitive mutants were obtained. Those mutants were again screened on the selective medium and five mutants, D33, D74, D154, D282, and D630 with enhanced resistance toward OA were obtained. Through TAIL-PCR, the flanking sequences were rescued. The sequences were blasted against TAIR database. The result indicated that the T-DNA of mutant D33 was inserted between At2g39720 (zinc finger) and At2g39730 (Rubisco activase) (GenBank accession No. EF591991), and the T-DNA junctions of the other four mutants were the same, all inserted in the same site of the first intron of At5g10450 (14-3-3 protein GF14 lambda) (GenBank accession No. EF591992).3. The expression of At2g39690,At2g39700, At2g39720 and At2g39750 were up-regulated in mutant D33 compared with wild type by semi-quantitation RT-PCR.4. The over-expression vectors of At2g39690,At2g39700 and At2g39720 were constructed and the transgenic plants were obtained by floral tip, and the resistance to S. sclerotiorum and OA will be detected among the transgenic plants.5. The putative OA-insensitive mutants were inoculated with agar plugs of S. sclerotiorum using detached leaves or in planta, mutants were more resistant than wild type. So the method to screen OA-resistant mutant, thus obtain S. sclerotiorum resistant mutant is practicable. It is possible to isolate the OA-resistant gene and obtain S. sclerotiorum resistant material.6. Oxalate decarboxylase (Yvrk) was cloned from Bacillus subtilis168, and oxalate oxidase was cloned from barley, and their plant expression vectors were constructed and transgenic plants were obtained by floral dip. The transgenic plants possess resistance to S. sclerotiorum. This result indicates a novel approach to develop transgenic plants resistant to fungal infection.7. Oligo microarray was used to investigate the differentially expressed genes under the stress of OA. The bioinformatics analysis indicated that OA induces the jasmonic/ethylene signaling, not salicylic acid signaling. The expressions of phenylpropanoid pathway, WRKY transcription family, cytochrome P450 family, ATP-binding cassette transporter family and heat shock family proteins were up- regulated, which indicating these pathway or family proteins were involved in the plant response to OA.8. MicroRNA microarray was used to investigate the differentially expressed micro-RNAs under the stress of OA. Three differentially expressed microRNAs and 9 predicted_miRNAs were obtained. Among them, one down-regulated microRNA is Ppt-miR1211 from Physcomitrella patens, and its target mRNA is At3g14520- terpene synthase based on miRU, another down-regulated microRNA is Ptc-miR3911 from Populus trichocarpa, which is identical to Ath-miR399b, and its target mRNA is At2g33770- a ubiquitin-conjugating E2 enzyme, both the two targets up-regulated in oligo-microarray. The only up-regulated differentially expressed microRNA is Ath-miR858, whose target mRNAs are At2g47460(DNA transcription factor)、At3g08500(DNA transcription factor)and At3g20310(ethylene responsive element- binding family protein. All the three target mRNAs were down-regulated in oligo-microarray, indicating the negatively-regulating function of microRNA.更多还原