【作者】 张慧娟；

【导师】 宋凤鸣；

【作者基本信息】 浙江大学 ， 植物病理学， 2009， 博士

【摘要】 植物的整个生长发育的过程就是和病原菌、环境相互适应、相互斗争的过程。在这个过程中,涉及了复杂的信号传导途径的协同作用。本研究中,我们分离克隆了水稻中与鞘氨醇-1-磷酸代谢相关的鞘氨醇激酶基因OsSPK1和OsSPK2及鞘氨醇-1-磷酸裂解酶基因OsSPL1、一个与受体类激酶同源基因OsBIK1,并对上述基因进行了功能分析,同时对拟南芥BIK1在磷营养调控中的作用进行了研究。鞘氨醇-1-磷酸（sphingosine-1-phosphate,S1P）在植物抗逆反应中起重要作用,但是在抗病反应调控中的作用仍然不清楚。为了研究S1P在植物抗病反应调控中的作用,分离鉴定了水稻S1P激酶基因OsSPK1和S1P裂解酶基因OsSPL1。RT-PCR分析表明,水杨酸、苯并噻二唑、ACC和茉莉酸等抗病信号分子处理能诱导OsSPK1的表达,但OsSPL1的表达略有下降;在水稻与稻瘟病菌的非亲和性互作反应中OsSPK1表达上调,但在亲和性互作中其表达没有明显变化。分别构建了过量表达水稻OsSPK1和OsSPL1的转基因烟草株系。抗病性测定表明,过量表达OsSPK1转基因植株提高烟草花叶病毒病（tobacco mosaic virus,TMV）和野火病（Pseudomonas syringae pv.tabacci）抗性,PR-1、PR-2、PR-3和PR-5等防卫基因表达上调,而过量表达OsSPL1的转基因植株降低TMV和野火病抗性。进一步比较了OsSPK1和OsSPL1转基因烟草植株的过敏性细胞死亡差异,发现注射20μM腐马霉素（fumonisin B1,FB1）处理后,在OsSPK1转基因植株叶片中没有观察到显著细胞坏死斑,而在OsSPL1转基因植株叶片上呈现出明显的过敏性细胞坏死斑。FB1处理前,相比于野生型植株叶片,OsSPK1转基因植株叶片中细胞死亡程度减轻,而OsSPL1转基因植株叶片中细胞死亡加重;FB1注射处理后,在OsSPK1转基因植株叶片中细胞死亡被抑制,而OsSPL1转基因植株叶片中细胞死亡加剧。这些结果暗示,由于过量表达OsSPK1或OsSPL1而改变S1P代谢后会引起转基因植株叶片中过敏性细胞死亡的发生。此外,过量表达OsSPK1转基因植株降低ABA敏感性,提高氧化胁迫,但抗盐性没有明显变化;而OsSPL1转基因烟草植株提高了ABA敏感性,降低氧化胁迫和抗盐性。这些结果表明,S1P代谢基因（或S1P本身）参与对抗病反应、过敏性细胞死亡以及抗逆反应的调控。AtBIK1编码一个受体样蛋白激酶,参与拟南芥对坏死性真菌病害抗性调控。但是,bik1突变体的根系在表型上类似于磷营养突变体。本文研究了BIK1是否参与调控拟南芥磷营养及其可能的机制。RT-PCR和P_BIK1::GUS分析表明,在缺磷条件下,AtBIK1被强烈地诱导;缺磷时,植株各个部位可以检测到GUS活性,虽然在正常磷营养条件下,侧根、主根、茎和部分叶片中检测到GUS活性,但缺磷条件下的活性更强。缺磷条件下,bik1突变体的侧根数量和长度、根毛数量和长度都比野生型显著增加,而主根生长并没有显著的区别。bik1植株中,叶片和根部中总磷含量增加,花青素含量升高,酸性磷酸酶活性降低,磷饥饿诱导基因的表达显著抑制。在缺磷的条件下,bik1植株内的过氧化氢量和超氧阴离子含量显著增加,miR399表达水平受到抑制。在正常磷和低磷条件下,Botrytis cinera和Alternaria brassicicola接种后,缺磷条件下bik1突变体植株上发病加快。这些结果表明,BIK1在植物磷营养中起重要作用,可能与miR399有关,而且磷营养状况影响到植物对坏死性真菌病害的抗性表型。拟南芥BIK1基因参与灰霉病抗病性调控。本文中,我克隆了水稻BIK1基因的同源基因OsBIK1。BTH、SA、ACC和JA等抗病信号分子能激活OsBIK1基因的表达,且在水稻与稻瘟病菌的非亲和性互作反应中OsBIK1基因上调表达。OsBIK1基因能互补拟南芥bik1突变体的功能。过量表达OsBIK1转基因水稻不仅长势比非转基因的植株好,提高了对稻瘟菌的抗性。此外,过量表达OsBIK1的转基因水稻植株增强了抗盐性、抗冻性以及抗干旱能力,但对ABA并没有反应。这些结果都表明了OsBIK1在水稻的抗逆、抗病和生长发育中起作用。更多还原

【Abstract】 During their life span,plants always suffer pathogen attack and environmental stress.To survive,plants have developed complicated and precisely regulated system to defend themselves against pathogens and abiotic stresses.To understand the molecular biology of defense responses in plants,I cloned and characterized genes involved in metabolism of sphingosine-1-phosphate(S1P),including OsSPK1, OsSPK2 and OsSPL1,and a receptor-like protein kinase gene OsBIK1,and performed functional analysis in transgenic tobacco and rice.Meanwhile,I also performed studies to elucidate the involvement of AtBIK1 in phosphorus nutrition in Arabidopsis.Recent studies have showed that S1P plays important role in regulation of abiotic stress responses.However,little is known about the role of S1P in regulation of defense response against pathogen infection.RT-PCR analysis showed that BTH (benzothiadiazole),SA(salicylic acid),ACC(1-aminocyclopropane-1-carboxylic acid) or JA(jasmonic acid,JA) can activate the expression of OsSPK1 and OsSPK2, but partially suppressed expression of OsSPL1.The expression of OsSPK1 and OsSPK2 was up-regulated in an incompatible interaction between Magnaporthe grisea and a resistant genotype,and no significant induced expression was observed in a compatible interaction.In order to investigate the role of S1P in plant disease resistance,functional analysis of OsSPK1 and OsSPL1 in transgenic tobacco plants were performed.Disease resistance assays revealed that overexpression of OsSPK1 in transgenie tobacco plants enhanced disease resistance against tobacco mosaic virus (TMV) and Pseudomonas syringae pv.tabaci(Pst),and up-regulated expression levels of defense-related PR gene,e.g.PR-1,PR-2,PR-3 and PR-5.Moreover, OsSPK1-overexpressing transgenic tobacco plants showed a reduced ABA sensitivity, and enhanced tolerance to oxidative stress.Significant necrotic lesion was observed in OsSPL1-overexpressing but not OsSPK1-overexpressing tobacco plants after injection with fumonisin B1(FB1),as revealed by detection of cell death in leaves by TUNNEL and staining approaches. It was previously shown that AtBIK1 plays important roles in defense response against necrotrophic fungi.Root architecture of the bik1 mutant seedling grown on normal MS medium,it was found that the root system of bik1 mutant is very similar to other mutants with defined function in phosphorous nutrition.RT-PCR and PBIK1::GUS analysis in transgenic lines demonstrated that AtBIK1 was expressed in the root,shoot and leaves in plants under low phosphorous condition.When in the phosphate deficient condition,the number and length of lateral root,the number and length of root hair were much more than the wild type.In bik1 mutants,it resulted in higher concentration of anthocyanin.In addition,when the mutants were deprived of phosphate,the activity of the total acid phosphatase was reduced while the total P content increased.Disease phenotypes of the wild-type and mutant both in the P sufficient and P deficient conditions were compared.The bik1 mutant in the P deficient condition was most susceptible to Botrytis cinera and Alternaria brassicicola,while the wild type in the P sufficient has the strongest disease resistance.These results suggest that the P condition affect the disease resistance. Together,these results suggest that AtBIK1 is a modulator of Pi starvation responses, including root development and the disease resistance.To investigate the function of OsBIK1,a functional analysis of OsBIK1 in transgenic rice plants was performed.RT-PCR analysis showed that BTH,SA,ACC and JA can activate the expression of OsBIK1.In the incompatible interaction between Magnaporthe grisea and a resistant genotype,OsBIK1 was strongly induced,while in the compatible interaction,there was no significant induction.Disease resistance assays revealed that overexpression of OsBIK1 in transgenic rice plants enhanced disease resistance against Magnaporthe grisea.Moreover,OsBIK1 overexpressing transgenic rice plants showed enhanced tolerance to cold stress,salt stress and drought. These results indicate that OsBIK1 play important role in defense responses against biotic and abiotic.更多还原