【作者】 狄翠霞；

【导师】 安黎哲；

【作者基本信息】 兰州大学 ， 植物学， 2008， 博士

【摘要】 多聚半乳糖醛酸酶抑制蛋白(poly-galacturonase inhibiting proteins,PGIPs)能与真菌分泌的内切多聚半乳糖醛酸酶(endo-polygalacturonase,PG)可逆专一性的结合,具有抑制真菌对植物细胞壁降解,激活植物的防卫反应,减缓真菌的繁殖速度等作用,因此被认为是一种植物抗病性相关防卫蛋白。本文以高山离子芥(Chorispora bungeana Fisch,et.Mey,Chorispora bungean)为实验材料,克隆到离子芥PGIP1(CbPGIP1)基因。通过免疫荧光分析了CbPGIP1在植株不同空间的分布。通过荧光定量PCR和Western blotting分析了生物胁迫和非生物胁迫下CbPGIP1基因的变化。此外,一氧化氮(nitric oxide,NO)在植物和病原菌互作中具有重要的重要。本文以拟南芥野生型(wild type)和一氧化氮合成酶突变体(Atnos1)为材料,研究真菌病原体胁迫下NO对植物抗病性以及AtPGIP1表达的调节机理。内容概括如下:1.从高山离子芥中克隆了CbPGIP1,此基因全长1203bp,编码332个氨基酸的蛋白质,分子量为36.9KD,等电点约为6.93。氨基酸序列分析表明,CbPGIP1蛋白中含有具有典型的LRR(leucine-rich repeat,富亮氨酸重复序列)特征。CbPGIP1与拟南芥AtPGIP1的同源性最高。因此命名为CbPGIP1。2.CbPGIP1和GFP融合蛋白通过基因枪转入洋葱表皮细胞的实验证实,CbPGIP1在细胞中没有特定的定位,均匀分布在细胞质中。3.荧光定量PCR和蛋白杂交证明茄匐柄霉,水杨酸(salicylic acid,SA)能够强烈的诱导CbPGIP1的表达,说明CbPGIP1在植物抗病中具有重要的作用。4.CbPGIP1的荧光免疫定位显示,在叶片中的CbPGIP1均匀分布在这个组织中,而茎和根中的CbPGIP1主要分布在表皮细胞和维管束中。进一步的实验显示CbPGIP1在叶中的含量高于在茎和根中的含量。这些结果显示CbPGIP1的空间分布做为一道天然的物理屏障抵御病源物的入侵。5.4℃,-4℃和NO均能诱导CbPGIP1基因的表达,CbPGIP1可能在非生物胁迫中具有重要作用。因为低温胁迫就像其他的胁迫条件一样能够降低植物的的抗病性。由此可见低温诱导CbPGIP的表达是为了增强植物的抗病性。NO诱导CbPGIP1基因表达说明了NO参与并调控了该基因的表达。6.荧光免疫定位显示,在拟南芥叶片中的AtPGIP1均匀分布在整个叶片组织中,而叶柄、茎和根中的AtPGIP1主要分布在表皮细胞和维管束中。这和离子芥的研究一致。AtPGIP1空间分布做为一道天然的物理屏障抵御病源物的入侵。7.NO增加植物对茄匐柄霉的抗性。T-DNA插入Atnos1基因的拟南芥突变体中,一氧化氮合成酶(nitirc oxide systhase,NOS)活性下降,NO释放减少。和拟南芥野生型相比,Atnos1突变体对于病原菌的生物胁迫更敏感。用NO供体SNP可以减缓Atnos1突变体对于病原菌的生物胁迫的敏感性。在植株水平上,Atnos1突变体在病原菌茄柄霉菌侵染后几乎不形成过敏性病斑。在组织水平上,病原菌茄匐柄霉在Atnos1突变体中繁殖更快。在细胞水平上,NO可以调控植物防卫反应中抗氧化酶含量。在信号分子水平上,突变体中游离态的SA含量比野生株的低。在基因水平上,突变体的一些抗病基因—AtPGIP1的表达量都比野生株的低。由此可见,NO作为信号分子,参与了对植物抗病胁迫的正调控。总之,病原菌,低温,SA和NO对离子芥PGIP诱导表明PGIP在植物的抗性反应中具有重要作用。同时,在植物的抗病防卫反应中,NO作为信号分子增强植物的抗病性并调控AtPGIP1基因的表达。更多还原

【Abstract】 Polygalacturonase-inhibiting proteins(PGIPs)are plant proteins believed to play an important role in the defence against plant pathogen fungals.PGIPs specifically inhibit fungal PGs,which reduce the hydrolytic activity of polygalacturonases(PGs),limit the growth of plant pathogens,and also elicit defence responses in plant.In our paper,PGIP gene was cloned from Chorispora bungeana(Chorispora bungeana Fisch,et.Mey,Chorispora bungean)(CbPGIP1). Immunofluorescence localizes the spatial distribution.Quantitative real-time PCR and western blotting characterized CbPGIP1 expression profile in response to biotic and abiotic stresses.In addition,Nitric oxide(NO)has emerged as a key molecule involved in the interaction between plant and pathogens.In our paper,the signaling functions of ON in plant fungal pathogen stress and the regulation of AtPGIP1 was studied using Arabidopsis thaliana wild type and Atnos1 mutant plants.The results were showed as following:1.The cloned full-length CbPGIP1 cDNA from Chorispora bungeana was 1203 bp.The cDNA encoded a protein of 332 amino acids with a calculated molecular weight of about 36.9 KD and with an isoeleetric point(pI)of 6.93. Prediction of amino acid analysis shows that the full-length cDNA of CbPGIP1 contains tepical LRR(leucine-rich repeat)domain.The protein exhibits closest homology to a subgroup of AtPGIP1 and we thus refer to it as CbPGIP1.2.CbPGI1P-GFP fusion protein was expressed transiently in epidermal cells of onions by microprojectile bombardment.The result showed that the intracellular distribution of fusion protein was located in cytosol,indicating no specific cellular localization of CbPGIP1 to any cellular compartment.3.Quantitative real-time PCR and western blotting showed that Stemphylium solani,SA(salicylic acid)notably induced the CbPGIP1 gene expression,indicating that CbPGIP1 plays key role in plant disease resistance.4.The results of immunofluorescence suggested that the CbPGIP1 in leaf was evenly localized in all tissues but the CbPGIP1 in stem and root was mainly localized in the epidermis and vascular bundle.Further study suggests that CbPGIP1 has higher levels in leaves and stems than in roots.Perhaps such impediments of the spatial distribution can be major factors as host resistance that places severe restrictions on the host range for a pathogen.5.4℃,-4℃and NO all can induce the CbPGIP1 gene expression,surgesting that CbPGIP1 would play important role in abiotic stresses.Low temperature like other stresses can increase susceptibility to diseases,so cold induction of CbPGIP1 might provide protection from infections.In addition,NO induced the expression of CbPGIP1,indicating that NO may take part in CbPGIP1 expression in Chorispora bungeana.6.The results of immunofluorescence showed that the AtPGIP1 in leaf was evenly localized in all tissues but the AtPGIP1 in petiole,stem and root was mainly localized in the epidermis and vascular bundle.This is consistent with the result of Chorispora bungeana.Those results indicate that the spatial distribution of AtPGIP1 as a physical barrier against the attack of fungal pathogen. 7.NO increased the plant resistance to Stemphylium solani.Atnos1 mutant was homozygous mutant line with a T-DNA insertion in the first exon of Atnos1 gene.NOS activity in Atnos1 was about 25%of wild type under normal condition. Atnos1 was identified to have impaired NO production.Compared with wild type plants,Atnos1 mutant plants were more sensitive to disease stress.Treatment of Atnos1 mutant plants with SNP(a NO donor)decreased the sensitive to disease stress.There were not hypersensitive response-like lesions in Atnos1 mutant plants infected by Stemphylium solani SS31.The spread of Stemphylium solani was more quickly in Atnos1 mutant plants than wild type plants.In addition,NO can regulate the antioxidative enzymes in plant defence.Compared with wild type plants,the extent of SA and AtPGIP1 were both lower in the mutant infected by Stemphylium solani.In a word,as a singal molecular,NO plays an important role in plant diease resistance.In a word,the induction of PGIP by infection,low temperature,SA and NO indicates that PGIP has a role in defence of subnival plant Chorispora bungeana.In addition,NO is a signal molecule involed in the enhancement of disease resistance and regulation of AtPGIP1 expression.更多还原