【作者】 鲍永美；

【导师】 张红生；

【作者基本信息】 南京农业大学 ， 应用植物基因组学， 2007， 博士

【摘要】 水稻是世界上最重要的粮食作物。在一些水稻产区,病原菌、高盐、干旱等逆境胁迫严重影响了水稻的生长和生产,因此阐明植物耐受逆境胁迫的分子机制和改良植物的抗逆性已经成为当前农业研究的主要目标之一。本论文的主要研究内容分为两个方面:一、从水稻中分离了4个与植物逆境胁迫信号传导途径相关的SNARE蛋白家族基因,并对它们的功能进行了初步研究;二、采用蛋白质组学方法分析了水稻抗稻瘟病质膜相关蛋白的诱导表达情况。真核生物细胞囊泡运输过程中的膜融合主要是由SNARE蛋白介导的,SNARE蛋白的结构高度保守。研究发现,植物中的SNARE蛋白促进植物细胞板形成,能与离子通道蛋白相互作用,有利于植物的正常生长发育,能提高植物的抗病性及参与植物的向重力性作用。本研究从水稻中克隆了水稻中的SNAP25类蛋白基因OsSNAP32,并对其功能进行初步研究。OsSNAP32基因编码蛋白含有283个氨基酸,在N端和C端分别存在一个Qb-SNARE结构域和一个Qc-SNARE结构域。洋葱表皮瞬时表达亚细胞定位结果表明,OsSNAP32基因的编码蛋白定位于细胞质膜上。半定量RT-PCR的结果表明,该基因的表达受到H₂O₂、PEG6000、SA、ABA、冷、热、稻瘟病菌（Magnaporthe grisea）和白叶枯病菌（Xanthomonas campestris pv.vesicator）这8种胁迫处理的诱导。对T₁代转基因植株进行稻瘟病菌接种处理,发现转正义OsSNAP32基因植株的抗瘟性与对照相比明显增强,转反义OsSNAP32基因植株的抗瘟性与对照相比无明显变化。综上研究结果表明OsSNAP32基因在水稻各种胁迫应答反应中起着重要的调节作用。本研究还克隆了Qb-SNARE蛋白家族成员基因OsNPSN11～13,这3个基因的序列同源性为80%-85%,具有相似的基因组结构,均含有10个外显子和9个内含子,分别定位于水稻第6、3和7条染色体上,多重序列比对结果表明这3个基因与拟南芥的AtNPSN11～13基因的同源性较高,所编码的蛋白均含有一个Qb-SANRE结构域和一个跨膜结构域。洋葱表皮瞬时表达亚细胞定位结果表明,这3个基因均定位于细胞质膜上。将OsNPSN11基因构建到表达载体pET-30a中进行原核表达,并进一步得到纯化蛋白制备多克隆抗体,用该抗体与水稻细胞的不同提取组分进行Western blot,结果表明该基因的编码蛋白定位于质膜上。为了研究水稻中OsNPSN11～13这3个基因的功能,我们用半定量RT-PCR的方法分析这3个基因在H₂O₂、NaCl和PEG6000处理下的表达情况,发现OsNPSN11～12基因在用H₂O₂进行处理时表达量均有所增加,而在用NaCl和PEG6000处理时,OsNPSN11-12基因的表达量有所下降,OsNPSN13基因的表达在各种处理情况下变化并不明显。进一步研究基因的功能,将这3个基因转入酿酒酵母细胞中,对转基因酵母细胞和对照酵母细胞进行H₂O₂、NaCl和甘露醇处理,结果发现,无论是在固体培养基上还是液体培养基上,在用H₂O₂进行处理时,转基因酵母细胞比对照酵母细胞长势好,而在用NaCl和甘露醇进行处理时,转基因酵母细胞比对照酵母细胞长势差表现为更加敏感。为进一步研究基因在植物中的功能,将基因转入烟草中,T₁代转基因幼苗在1/2MS培养基上进行H₂O₂、NaCl和甘露醇处理,结果发现H₂O₂处理后,转基因烟草比未转基因对照烟草长势好,用NaCl和甘露醇处理后,转基因烟草比未转基因对照烟草长势差。由上可知,OsNPSN11～13可能参与酵母和烟草的抗H₂O₂胁迫过程,而在NaCl和甘露醇胁迫过程中可能不起作用。进一步分析OsNPSN11～13这3个基因在抵御生物胁迫过程中的功能。通过半定量RT-PCR的方法分析了水稻植株在接种白叶枯病菌和稻瘟病菌处理后的表达情况发现,在白叶枯病菌接种处理后,OsNPSN11～12基因的表达受到诱导,在稻瘟病菌接种处理后,抗病品种黑壳子粳中OsNPSN11～12基因的表达受到诱导,感病品种苏御糯中OsNPSN11～12基因的表达在处理前后没有明显变化,OsNPSN13基因的表达在两种病原菌处理前后均没有明显变化。鉴于OsNPSN11～12基因在抗稻瘟病品种和感病品种中的表达差异,我们将OsNPSN11基因转化感病品种苏御糯受体,对T₀代转基因植株进行PCR、RT-PCR和Southern blot检测得到阳性植株后,对T₁代转基因植株进行苗期抗瘟性鉴定,结果发现转正义OsNPSN11基因植株的抗性明显增强。以上实验结果表明,OsNPSN11基因在水稻植株抗病过程中起着一定的作用,具体的抗病机制有待进一步的实验证明。植物质膜蛋白在植物抵御病原物侵染早期信号传导途径中起着重要的作用。本文采取改进的质膜蛋白提取、纯化和双向电泳方法,分析了太湖流域地方抗病品种黑壳子粳在稻瘟病菌菌株北1接种处理前后质膜相关蛋白的诱导表达情况。比较分析了未处理和接种处理8h和24h后质膜蛋白的双向电泳图谱,发现有23个蛋白点受到了不同程度地诱导,并对这23个蛋白点进行了质谱鉴定,其中7个蛋白点进行二级串联质谱（MS/MS）结果表明这23个诱导表达蛋白可分为四类:1)与植物生长代谢相关的酶类,如与植物糖酵解相关的丙糖磷酸异构酶（TIM）、6-磷酸葡萄糖酸脱氢酶（6PGDH）和3-磷酸甘油醛脱氢酶（GAPD）,与脂类代谢相关的烯酰CoA水合酶（ECH）,与能量代谢相关的苹果酸脱氢酶（MDH）、乙醇脱氢酶（ADH）、1,4苯醌还原酶（QR）和苯醌氧化还原酶（QR2）,与氨基酸代谢相关的甲酰四氢叶酸合成酶（FTHFS）;2)与植物物质合成分解调节有关的蛋白,如调控质膜多肽（DREPP）、可逆糖基化多肽（RGP）、Remorin蛋白和20S蛋白酶体;3)与抗氧化系统相关的蛋白,如脱氢抗坏血酸还原酶（DHAR）、超氧化物岐化酶（SOD）、抗坏血酸过氧化物酶（APX）;4)与信号传导有关的蛋白,如膜连蛋白（Annexin）和谷胱甘肽转移酶（GST）。本研究发现了一些新的抗病防卫反应早期响应质膜相关蛋白,为了解水稻抗稻瘟病防卫反应机理提供了新的线索。更多还原

【Abstract】 Rice is the most important staple crop that is cultivated worldwide.In the many rice-growing areas there are many pathogens like fungi,bacteria or viruses,frequent salinity and extreme drought to impede rice growth and production.It has been one of main objects for the researchers to elucidate the mechanism of plant resistance or tolerance to a variety of stresses and improve the ability of crops to resistance to the stresses.This thesis aims to do functional analysis of four SNARE protein genes from rice and proteomic analysis of rice blast defense responsive plasma membrane associate proteins,and main results are given as follows.The membrane fusion in vesicle trafficking in the cells of eukaryotic organisms is mediated by soluble-N-ethyl-maleimide-sensitive fusion protein attachment protein receptor（SNARE） proteins,which are highly conserved from various species.Increasing research has demonstrated SNAREs involved in the formation of the cell plate,interacting with ion channel proteins,and gravity sensing in plants.SNARE proteins might play important roles in plant growth,development and response to abiotic and biotic stresses. Here we report the cloning and expression characterization of OsSNAP32 from rice,a novel member of SNAP25-type proteins.The OsSNAP32 comprises 283 amino acid residues with the molecular weight of 31.3kD,and contains Qb-SNARE and Qc-SNARE domains in the N- and C-terminal.By the onion epidermal cells transient expression method,it revealed that OsSNAP32 had the plasma membrane located character of SNAP25-type proteins.Semi-quantity RT-PCR assay showed the expression of OsSNAP32 was significantly activated in rice seedlings treated by H₂O₂,PEG6000,SA,ABA,low temperature and hot or inoculated by rice blast（Magnaporthe grisea） and blight （Xanthomonas campestris pv.vesicator）.The T₁ OsSNAP32 over-expressing transgenic plants seemed increased resistance ability to rice blast.The results suggested that this gene may be belong to a novel gene family encoding SNAP25-type proteins involved in the rice responses to biotic and abiotic stresses.In additon,we report three novel plant SNARE（NPSN） genes isolated from rice and named OsNPSN11,OsNPSN12 and OsNPSN13.They have 80-85%nucleotide identity and similar genomic organization,with ten exons and nine introns in each gene.OsNPSN11, OsNPSN12 and OsNPSN13 are located on chromosomes 6,3 and 7,respectively.Multiple alignment of deduced amino acid sequences indicate that the OsNPSN11～13 proteins are homologous to AtNPSNs from Arabidopsis,containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region.Transient expression analysis in onion epidermal cells revealed that OsNPSN11～13 were located on the plasma membrane. The cDNA of OsNPSN11 was subcloned into pET-30a,and the recombinant protein expressed in E.coli was obtained.With the polyclonal antibody made by the purified protein, western blot result showed that OsNPSN11 was located on the plasma membrane in rice cells.Semi-quantitative RT-PCR assays showed that the expression of OsNPSN11～12 were significantly activated in rice seedlings treated with H₂O₂,but decreased under salt and PEG6000 stresses.Transformed yeast cells with OsNPSN11～13 had better growth rates than wild type or empty vector transformants when cultured in solid or liquid media containing various concentrations of H₂O₂.However the transformed yeast cells with OsNPSN11～13 were more sensitive to salt and mannitol stresses.The transgenic tobacco with OsNPSN11～13 grow better than wild type when treated with H₂O₂,while showed more sensitive to salt and mannitol treatments.These results indicate that the products of OsNPSN11～13 may be involved the signal transduction in yeast and tobacco responses to abiotic stresses.We also study about the function of OsNPSN11～13 under biotic stresses.Semi RT-PCR results showed that the expression of OsNPSN11～12 in variety Heikezijing were induced by rice blight and rice blast incubation,however,the expression of OsNPSN11～12 in variety Suyunuo were unchanged by rice blast incubation.According to the different expression pattern between the varieties Heikezijing and Suyunuo,we transformed OsNPSN11cloned from Heikezijing to Suyunuo.After identification by PCR,RT-PCR and Sourthern blot,the T₁ OsNPSN11 over-expressing transgenic rice seedlings also showed increased disease resistance ability.These results indicated the roles of OsNPSN11 in disease response of rice.Plant plasma membrane（PM） proteins play important roles in signal transduction during defense response to an attacking pathogen.By using an improved method of PM protein preparation,purification and 2-D gel analysis,we conducted PM proteomic analysis of the rice Heikezijing leaves to identify PM components involved in the early defense response to rice blast fungi（Magnaporthe grisea Hoku 1）.A total of 23 regulated protein spots were observed on 2-D gels of PM fractions at 8 and 24 h after pathogen inoculation, of which were induced with different levels.23 protein spots with predicted functions in plant defense were identified by MS,including 7 MS/MS identified protein spots.All of the induced proteins can be divided in to four types:first,associate with metabolizing,such as triosephosphate isomerase（TIM）,6-phosphogluconate dehydrogenase（6PGDH）, glyceraldehyde-3-phosphate dehydrogenase（GAPD）,enoyl-CoA hydratase（ECH）,malate dehydrogenase（MDH）,alcohol dehydrogenase（ADH）,1,4-benzoquinone reductase（QR）, quinone oxidoreductase（QR2） and formyltetrahydrofolate synthetase（FTHFS）;second, associate with regulating peptide,as developmentaly regulated plasma membrane polypeptide（DREPP）,reversibly glycosylated polypeptide（RGP） and 20S proteasome subunit;third,associate with detoxification enzymes,such as ascorbate peroxidase（APX） and Cu/Zn superoxide dismutase（SOD）;fourth,associate with signal transduction,such as annexin and glutathione-S-transferase（GST）.Our study would provide a starting point for functional research of PM proteins in the rice defense.更多还原