【作者】 刘丽华；

【导师】 王宗华； 汪世华；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 稻瘟病菌(Magnaporthe oryzae)引起水稻重要病害,是研究植物与病原微生物相互作用的重要模式生物。已知其在侵染水稻时分泌几丁质酶、丝氨酸蛋白酶,参与致病过程。前期研究表明稻瘟病菌几丁质酶和丝氨酸蛋白酶可能是稻瘟病的致病因子,然而,它们在病程中的作用机制未明,特别是如何介导寄主水稻的响应,尚无报道。本文利用酵母双杂交系统以稻瘟病菌几丁质酶、丝氨酸蛋白酶作为诱饵蛋白,从水稻中筛选受体,研究两个蛋白与候选受体的互作关系,并探讨候选受体水稻RNAi突变体在与稻瘟病菌互作过程的变化,从而进一步明确它们在抗病或致病过程中的作用。酵母双杂交获得与稻瘟菌几丁质酶(MoChi1)互作的阳性克隆18个,与稻瘟病菌丝氨酸蛋白酶(MoSP1)互作的阳性克隆12个,同时测序并对他们进行生物信息学分析,结果发现MoChi1和MoSP1筛选到了同一个受体甘露糖凝集素OsMbl1。通过GST pull-down验证了MoChi1可以与水稻甘露糖凝集素OsMbl1、金属硫蛋白OsMtl1和MoSP1与锌指蛋白OsZfp1、甘露糖凝集素OsMbl1及A11体外互作。Western bloting结果表明MoChi1与OsMbl1和OsMtl1间存在相互作用;MoSP1可以与OsZfp1、OsMbl1体外互作,但不能与A11体外互作。本文重点研究了OsMbl1分别与MoChi1和MoSP1互作结构域。首先,生物信息学分析表明,OsMBL1含有单子叶植物甘露糖结合凝集素家族结构域,且该结构域含有三个糖结合位;与蒜、石斛、文殊兰属等其他单子叶植物甘露糖结合凝集素的序列一致性大于50%,并具有单子叶植物甘露糖结合凝集素保守酸性氨基酸的结合位点。通过构建OsMBL1不同片段缺失突变体,经GST pull-down方法来检测其与MoChi1和MoSP1的互作特性,结果表明在与几丁质酶互作的过程中他的三个结合位点G…..GXXXD、GXGXXXEDE和GX[GAVIYWF][DNEW]是相互独立的,都能各自发挥作用。然而它与丝氨酸蛋白酶互作结果却表明只有这三个位点同时存在时才能表现出活性。本文还分析了MoSP1与OsZfp1的互作结构域。生物信息学分析表明,OsZFP1含有一个C3HC4保守结构域,C端位置有两个跨膜螺旋序列,编码α/β型、非分泌型蛋白。缺失突变和GST Pull-down分析表明,OsZfp1与丝氨酸蛋白酶的互作只需要其C3HC4结构域,其N端和C端不是它们互作所必须的。利用实时定量PCR技术检测OsZFP1、A11和OsMBL1基因在几丁质酶和丝氨酸蛋白酶敲除突变体和野生型Guy11侵染后的水稻叶片中的表达情况,结果表明,在丝氨酸蛋白酶敲除突变体侵染后的水稻叶片中表达量与在野生型侵染后的水稻叶片中的表达量相比,OsMBL1在72小时前表现为下调,在96小时后表现为上调;OsZFP1都表现为下降;而A11基因的表达量无多大差异。OsMBL1基因在几丁质酶敲除突变体侵染后的水稻叶片中的表达量先表现为下调,48小时后达到最小,接着又有所回升。与野生型侵染后的水稻叶片中的表达量相比,在72小时前表现为下调,在96小时后表现为上调此外,本研究还分别构建了OsMBL1和OsZFP1基因的RNAi及超表达载体并转化水稻,目前已获得OsMBL1和OsZFP1基因的RNAi植株,超表达转基因植株尚未获得。因而,两个蛋白的生物学功能还有待深入研究。更多还原

【Abstract】 The rice blast fungus, Magnaporthe oryaze, infects many economically important cereal crops, particularly rice. The interaction between M. oryzae and rice is also taken as a model for the study of fungus-plant interaction. It has been shown that the fungus could secrete chitinase and serine protease in the process of its host infection. Our previous studies indicated that chitinase and serine protease in M. oryaze are probably important pathogenic factors. However, their regulation mechanism in the process of blast fungus infection remains unclear, particularly in the regulation of pathogen-associated molecular pattern (PAMP)-induced immunity reaction. In this study, we characterized the relationship between the chitinase/serine protease in M. oryzae and their receptors in rice by the yeast two-hybrid assay, immunology and molecular genetics approach by creating the rice RNAi mutants of putative receptors.By yeast two-hybrid screening, we obtained 18 rice positive clones interacting with M. oryzae chitinase (MoChi1) and 12 rice positive clones interacting with M. oryzae serine protease (MoSP1). These clones were then sequenced and annotated by BLAST search. Interestingly, we found a common receptor, mannose-binding lectin (OsMbl1), which could interact with both MoChi1 and MoSP1.GST pull-down and Western bloting were then to confirm interaction between above candidates and MoChi1 and MoSP1. Results showed that there is interaction between the mannose-binding lectin, metallothionein-like protein (OsMtl1) and MoChi. And there was an obvious interactrion between OsMbl1, OsZfp1 and MoSP1. We then further dissected the binding properties of a protein-protein interaction domain between OsMbl1 and MoChi1 and MoSP1. First, bioinformatics analysis showed that OsMBL1 contains mannose-binding lectin domain, including three sugar-binding sites. Homology analysis showed that OsMbl1 is about 50% identical to other mannose-binding lectin from Monocotyledon, such as garlic, dendrobium and crinum. And it has conserved amino acid binding sites of mannose-binding lectin of Monocotyledon. In order to clarify the interaction domain of the smallest fragment between OsMbl1 and MoChi1 and MoSP1, we constructed the different mutants of OsMBL1, then detected the interaction by GST Pull-down assay. The results showed that the three binding sites, G…..GXXXD,GXGXXXEDE and GX[GAVIYWF] [DNEW], are independent for each other during their interaction with MoChi1. Whereas, the three binding sites are dependent for each other during their interaction with MoSP1.We also analyzed the interaction domain of another receptor OsZfp1 with MoSP1 in M. oryzae. Bioinformatics analysis indicated that the OsZFP1 gene contains a C3HC4 conserved domain, two c-terminal transmembrane helix sequence, which coding andα/β-type, non-secreted protein. Domain deletion and GST Pull-down assay demonstrated that N-terminal and C-terminal of this protein is not necessary for its interaction with the serine protease, while only C3HC4 domain is required for the interaction.The expression of OsZFP1, A11and OsMBL1 mRNA in the rice leaves which were infected by Guy11 or the knockout mutant of MoChi1 and MoSP1 were examined by the Real-Time PCR. The results showed the expression of OsMBL1 mRNA in the rice leaves infected by the knockout mutant of serine protease was down-regulated before 72 h compared with that in the Guy11-infected rice leaves,the expression of OsZFP1 was also down-regulated. However, the expression of A11 mRNA had no difference in all mutants. The expression of OsMBL1 mRNA in the rice leaves infected by the knockout mutant of MoChi1 was down-regulated compared with that in the Guy11-infected rice leaves., which is consistent with the results above.Besides above results,we also plan to analyze the biological functions of OsZFP1 an OsMBL1 through RNAi and overexpression in the rice. We have obtained RNAi and overexpression constructs of these two genes and the transgenic rice of RNAi-OsZFP1 and RNAi-OsMBL1. The lines of overexpression-OsZFP1 and overexpression -OsMBL1 were not obtained yet. Therefore, more detailed analysis of the transgenic lines generated from this study are necessary for further identification the function of these genes in rice.更多还原