【作者】 李国钧；

【导师】 宋凤鸣；

【作者基本信息】 浙江大学 ， 植物病理学， 2010， 博士

【摘要】 聚ADP-核酸化是一类在哺乳动物系统中起重要作用的转录后蛋白修饰方式。最近研究表明,聚ADP-核酸化也参与了植物的生长发育调节和逆境反应。本文利用T-DNA插入突变体和过量表达转基因株系研究了拟南芥聚ADP-核酸葡聚糖水解酶1[poly(ADP-ribose)glycohydrolase 1, PARG1]基因在灰霉病(Botrytis cinerea, B. cinerea)抗性和非生物逆境反应中的作用。RT-PCR分析结果表明,B. cinerea侵染拟南芥,植株叶片中PARG1转录水平逐渐增强；光照诱导拟南芥植株叶片中PARG1转录水平增强；黑暗条件下,以水杨酸(salicylic acid, SA)喷雾处理拟南芥植株,SA诱导拟南芥植株PARG1转录水平增强,但在处理SA时遇光照,PARG1基因下调表达。茉莉酸(jasmonic acid, JA)、乙烯前体1-氨基环丙烷-1-羧酸(1-aminocyclopropane-1-carboxylic acid, ACC)不诱导PARG1基因的表达。35S-GFP::PARG1结果显示,PARG1定位于细胞核和细胞质中。构建了PPARG1::GUS转基因株系,染色结果显示,PARG1在拟南芥植株的根尖、叶、花药内表达。为了研究PARG1在抗病反应中的功能,筛选获得一个基于Ws-0生态型背景的T-DNA插入突变体parg1-3和一个基于Col-0生态型背景的T-DNA插入突变体parg1-4;构建了由CaMV 35S启动子驱动的基于parg1-3突变体背景的转PARG1基因过量表达系,PARG1-OE1/parg1-3和PARG1-OE2/parg1-3,与基于野生型Col-0背景的转PARG1基因过量表达系PARG1-OE1/Col-0和PARG1-OE2/Col-0。接着比较分析了上述突变体和过量表达转基因株系接种B. cinerea和番茄细菌性斑点病菌(Pseudomonas syringae pv. tomato DC3000, Pst DC3000)的病害表型。喷雾接种B. cinerea或注射接种Pst DC3000后,parg1突变体的病害发病水平轻于野生型的病害发病水平,野生型的病害发病水平则轻于PARG1-OE株系的病害发病水平。接种病原菌后做菌量测试,parg1突变体叶片中B. cinerea生长量和Pst DC3000菌量均低于野生型中相应病菌菌量,野生型叶片中B. cinerea生长量和Pst DC3000菌量均低于PARG1-OE株系叶片的相应病菌菌量。B. cinerea侵染后,各株系病程相关蛋白基因(Pathogenesis-related protein gene,PR.)基因表达水平均无显著差异,说明PARG1调控拟南芥对B.cinerea抗病性不是通过调控PRs基因表达实现的。考虑到活性氧和细胞死亡在B.cinerea侵染植物过程中的作用,以B.cinerea接种拟南芥,分析野生型、pargl和PARG1-OE株系的活性氧产生和细胞死亡状况。发现pargl突变体叶片中积累的活性氧含量比野生型叶片积累的活性氧含量少,而野生型叶片中活性氧的积累比PARG1-OE株系叶片中积累得少；同时发现在B.cinerea发病过程中,parg1植株叶片细胞的死亡数量比野生型叶片细胞的死亡数量少。为了进一步研究PARG1基因在细胞死亡中的功能,分别对拟南芥各株系接种Pst DC3000(avrRPM1)和高浓度Pst DC3000,PARG1-OE株系叶片细胞均比野生型叶片细胞易于死亡,而野生型叶片细胞均比parg1突变体叶片细胞易于死亡,说明PARG1参与过敏性细胞死亡的调控。考虑到聚ADP-核酸[poly(ADP-ribose),PAR]作为死亡信号的作用,应用PAR处理野生型、parg1突变体和PARG1-OE株系,发现PAR能诱导parg1突变体细胞凋亡,但并不引发细胞坏死,而caspase-3抑制剂Ac-DEVD-CHO能部分抑制PAR引发的细胞凋亡,表明受PARG1调控的胞内PAR能诱导细胞凋亡,其凋亡途径部分依赖caspase凋亡途径。本文还比较分析了parg1-3突变体和转基因过量表达株系对干旱等非生物逆境的表型差异。基因表达结果表明,干旱和氧化胁迫处理,野生型拟南芥中的PARG1基因上调表达。在含甘露醇和甲基紫腈(methyl Viologen,MV)的培养基中进行种子萌发实验,模拟干旱和氧化胁迫逆境,发现parg1-3种子的发芽率比野生型种子发芽率降低,但以脱落酸(absisic acid,ABA)处理进行萌发实验,两者种子表现出相似的发芽率。在干旱、渗透和氧化胁迫下,parg1-3植株比野生型植株表现出较弱的忍耐力。在干旱逆境下,野生型植株叶片的气孔完全闭合,而parg1-3植株叶片的气孔不能完全闭合；但在ABA处理下,parg1-3植株叶片与野生型植株叶片的气孔闭合状况相似。此外,在干旱逆境中,parg1-3植株与野生型植株的干旱逆境／ABA反应基因表达水平相似。而在氧化胁迫下,parg1-3植株中的氧化胁迫相关基因,如交替氧化酶1(alternative oxidase 1,Aox1)和抗坏血酸过氧化物酶(ascorbate peroxidase 2,Apx2)下调表达。不论是干旱、渗透、氧化胁迫处理,还是ABA处理,PARG1的转基因过量表达株系均未表现出与野生型植株的差异。这些结果表明,PARG1基因在拟南芥对多种类型的逆境反应中起重要作用。上述结果表明,PARG1基因可能通过调节细胞内的PAR含量,维持细胞内NAD+和ATP含量,在植物抗病和抗逆反应中起重要作用。研究结果对全面认识和理解拟南芥在抗病、逆境反应中的分子机制以及高等植物中聚ADP-核酸化的作用有积极的意义。更多还原

【Abstract】 Poly(ADP-ribosyl)ation is a post-translational protein modification that plays important roles in many cellular processes in mammal systems. Emerging evidence indicates that poly(ADP-ribosyl)ation is also involved in regulation of growth/development, stress and defense response in plants. In this study, I studied the possible function of Arabidopsis poly(ADP-ribose) glycohydrolase 1 (PARG1) in disease resistance and abiotic stress responses using T-DNA insertional mutant and overexpression transgenic lines.Bioinformatics analysis reveals that there are two genes encoding for poly(ADP-ribose) glycohydrolases. RT-PCR analysis showed that expression of PARG1 was up-regulated in Arabidopsis plants after inoculation with Botrytis cinerea(B. cinerea), a necrotrophic fungal pathogen. Similarly, expression of PARG1 was also significantly induced in Arabidopsis plants after treatment with salicylic acid (SA) or light. By contrast, the expression of PARG1 was reduced when Arabidopsis plants was treated with SA under the light. Expression of PARG1 in Arabidopsis was not be induced after treatment with jasmonic acid (JA) and 1-aminocyclopropane-l-carboxylic acid (ACC). PPARG1::GUS transgenic lines was generated and GUS staning showed that the PARG1 gene was expressed in root tips, leaves and anthers. Subcellular localization analysis using 35S-GFP::PARG1 showed that the PARG1 protein was localized in the nucleus and cytoplasm of Arabidopsis cells.To study the function of PARG1 in disease resistance and abiotic stress responses, two T-DNA insertion line, parg1-3 in Ws-0 background and parg1-4 in Col-0 background were identified from genotyping screening. Meanwhile, transgenic lines overexpressing the PARG1 gene, driven by CaMV 35S promoter, in pargl-3 mutant and wild type Col-0 background were also generated. Homozygous lines with single copy of the PARG1 transgene were obtained through antibiotic resistance segregation screenings. Two independent transgenic lines overexpressing PARG1 gene in parg1-3 mutant background (PARG1-OE1/parg1-3 and PARG1-OE2/parg1-3) or wild type Col-0 background (PARG1-OE1/Col-0 and PARG1-OE2/Col-0) were selected for further studies. Disease phenotypes of the wild-type, pargl and PARG1-OE plants were compared after inoculation with B. cinerea or Pseudomonas syringae pv. tomato DC3000(Pst DC3000). As compared with the wild-type plants, the pargl plants showed increased resistance to B. cinerea and Pst DC3000. However, the transgenic PARG1-OE plants showed significant increased susceptibility to the above-mentioned pathogens. Furthermore, growth of B. cinerea and Pst DC3000 in leaves of the pargl plants was much less than in the wild-type plants; whereas growth of B. cinerea and Pst DC3000 in leaves of the PARG1-OE plants was significantly increased. After inoculation with B. cinerea, expression of Pathogenesis-related protein gene(PR) was similar in the wild-type, pargl and PARG1-OE plants. These results indicate that PARG1-mediated defense response against B. cinerea may be independent on PR genes. These data suggest that PARG1 plays an important role in Arabidopsis defense responses against both necrotrophic and biotrophic pathogens.To further study the function of PARG1 in disease resistance response, we examined accumulation of reactive oxygen species and levels of apoptosis in Arabidopsis after inoculation with B. cinerea. It was observed that, after B. cinerea infection, PARG1-OE plants accumulated much more reactive oxygen species in leaves but the pargl plants accumulated less reactive oxygen species in leaves, when comopared with those in the wild-type plants. As compared with the wild-type plants, the pargl plants showed reduced level of apoptosis after B. cinerea infection, whereas the PARG1-OE plants showed accelerated cell death after inoculation with B. cinerea, Pst DC3000 (avrRPM1) or Pst DC3000. these results suggest that PARG1 may be also involved in regulation of cell death. Poly(ADP-ribose)(PAR) induces apoptosis in pargl plants, but did not induce cell necrosis. Ac-DEVD-CHO, a caspase3 inhibitor, inhibited partially PAR- induced apoptosis. These results demonstrate that PAR, which regulated by PARGl, can induce apoptosis, and this process is partially dependent on caspase-apoptosis signaling pathways.I also examined the possible function of PARG1 in Arabidopsis abiotic stress tolerance using genetic mutant parg1-3 and transgenic PARG1-overexpressing plants. Expression of PARG1 in wild type plants was upregulated in response to drought and oxidative stress treatments. Germination rates of the parg1-3 seeds were reduced under mannitol or methyl viologen (MV) treatment as compared with the wild type seeds, but the parg1-3 seeds showed similar germination rate to the wild type seeds under ABA treatment. The parg1-3 plants showed reduced tolerance to drought, osmotic and oxidative stresses, with increased levels of cell damage under osmotic and oxidative stress and reduced survival rate under water-withholding drought stress, as compared with the wild type plants. Stomatal apertures of parg1-3 plants failed to close under water-withholding drought stress, but showed similar closure patterns to the wild type plants after ABA treatment. As compared with those in wild type plants, drought stress/ABA-responsive genes in parg1-3 plants showed comparable expression levels under drought stress condition, whereas expression level of some of oxidative stress-related genes in parg1-3 plants was reduced after MV treatment. Overexpression of PARG1 in parg1-3 mutant and wild type Col-0 background did not cause any phenotypical changes in response to drought, osmotic and oxidative stresses and ABA treatment. These results suggest that PARG1 plays important roles in abiotic stress tolerance.Taken together, data presented in this thesis suggest that PARG1 plays important roles in disease resistance and abiotic stress responses.更多还原