【作者】 韩成云；

【导师】 任春梅； 谢道昕；

【作者基本信息】 湖南农业大学 ， 遗传学， 2012， 博士

【摘要】 模拟病斑突变体(lesion mimic mutant)是一类在没有明显的逆境、损伤或病原物侵害时,在叶片上能自发地形成类似病原物侵染后的坏死斑的突变体,这些突变体往往能激活植物体的系统获得性抗性,通常具有与抗病反应相关的细胞学和生物化学特征。拟南芥短日照依赖型模拟病斑突变体(short-day-dependent lesion mimic mutant1, sdl1),是一个在短日照条件下(8小时光照、16小时黑暗)形成模拟病斑的突变体。该突变体在长日照条件下(16小时光照、8小时黑暗)与野生型拟南芥(Col-0)没有明显差异；但在短日照条件下,生长减慢,个体较野生型矮小,叶片局部产生类似坏死的病斑。植株抽苔后,幼嫩的茎以及茎顶端的花序和果荚会发生白化萎蔫,但不导致植株整体死亡。进一步研究突变体的表型,发现生长在培养基上的sdl1突变体仍然表现模拟病斑坏死表型,培养基中高浓度蔗糖的添加在一定程度上削弱了模拟病斑坏死的发生。采用图位克隆的方法分离鉴定了SDL1基因,该基因编码含421个氨基酸的蛋白质。sdl1的单碱基突变位点发生在基因第四个外显子的最后一个碱基,直接导致编码的氨基酸序列第157个氨基酸由Trp变为终止密码子TAG,使翻译提前结束,丢失了265个氨基酸。在模拟病斑形成的过程中,大多数突变体都表现出与病原菌防御相关的组织化学及系统获得抗性相关基因的表达。本研究将各生长时期的叶片进行台盼蓝染色,发现均有不同程度的细胞死亡。检测突变体中部分病程相关基因的表达,发现在短日照条件下,sdl1突变体激活了水杨酸信号途径的抗病基因表达,并伴随内源水杨酸的积累；通过构建sdl1与水杨酸缺失突变体NahG及水杨酸信号转导缺失突变体npr1-1的双突变体,发现模拟病斑的产生与水杨酸在植物体内的积累密切相关,而与水杨酸的信号转导关系不大。由于突变发生在剪接识别位点,采用cDNA测序及RT-PCR检测发现没有发生剪切位点的改变。RT-PCR分析发现sdll突变体中的SDL1基因的表达显著降低。本研究进一步通过构建含有内源启动子的表达载体以及由35S强启动子启动的超表达载体来检测基因mRNA及蛋白水平的表达。结果表明SDL1基因剪接识别位点的突变并没有影响剪切效率,基因通过启动子自身进行反馈调控。此外,mRNA及蛋白水平表达说明了SDL1基因的表达不受光照的调控。SDL1是一个未经报道的新基因,生物信息学分析表明,SDL1高度保守,具有延胡索酰乙酰乙酸酶(fumarylacetoacetate hydrolase, FAH)重要结构域,推测其可能作为FAH参与了植物体内酪氨酸的代谢进程。本研究借鉴动物体内的Tyr代谢模式,外源添加芳香族氨基酸及其代谢中间产物和终产物来处理植物,进一步寻找基因作用的节点。研究发现,外源添加高浓度的Tyr及Trp可以抑制sdl1突变体在短日照条件下的模拟病斑表型；而外源添加Phe对sdl1突变体几乎没有影响。氨基酸分析发现sdl1突变体中Tyr的含量没有改变。说明一定量的Tyr及Trp的添加似乎可以弥补SDL1基因突变的影响。低浓度的代谢中间产物尿黑酸(homogentisate, HGA)加速了sdl1突变体的模拟病斑坏死表型的发生,而高浓度HGA强烈抑制植物生长。代谢终产物延胡索酸(fumaric acid, FA)的添加没有影响,但SDL1基因突变降低了植物体内的FA含量。说明SDL1基因很可能是在HGA的下游,FA的上游发挥作用,但这种表型并非是由于HGA的积累及FA减少带来的。综上所述,本文分离鉴定了拟南芥短日照依赖型模拟病斑SDL1基因,并对该基因的功能进行了初步探讨,研究结果对探讨光照长度如何调控植物抗病性的分子机理具有重要的科学意义。更多还原

【Abstract】 Lesion mimic mutants spontaneously develop lesions without apparent adversity, injury or pathogens. The lesion formation seems to be similar to the hypersensitive response elicited by inoculation with an avirulent pathogen. In some cases, lesion mimic mutants express cytological and biochemical markers that have been associated with disease-resistant responses, and exhibit local and systemic acquired resistance (SAR).A mutant which develops lesions under short-day conditions(8hr light/16hr dark) was named as short-day-dependent lesion mimic mutant1, sdl1. The sdl1mutant shows no significant differences with the wild-type Col-0under long-day conditions (16hours light/8hours dark), but the mutant plant grows more slowly and is more dwarf than the wild-type plant under short-day conditions. The sdl1mutant spontaneously develops lesions on its leaves without pathogen attack and wrinkles irregularly. After bolting, the acrocarpous plant shows albino stems and wilting siliques and flower buds. But it will not lead to the death of the mutant. Further studies reveal that the lesion phenotype of sdl1could not be suppressed in medium plates, but was able to be obviously attenuated by supplementation of high concentration sucrose in medium.The SDL1gene is identified by map-based cloning. Arabidopsis SDL1gene encodes a421amino acid protein. The mutation in sdll occurs at the last base of the fourth exon of SDL1gene, which directly leads to a substitution of the termination codon TAG for Trp. The translation ends at a premature end, losing265amino acids.In the process of mimic lesion formation, it has been detected in most of the lesion mimic mutants that the pathogen defense-related histochemical reaction and the systemic acquired resistance-related gene expression. Partial cell death has also been observed through the trypan blue staining in the leaves’cells of different growth periods. By detecting the resistance genes expression level, we discovered the constitutive expression of related resistance genes and the accumulation of salicylic acid in the mutants under short-day conditions. It suggests that the lesion mimic formation is related to the accumulation of SA in the mutants, but unrelated to SA signaling pathways by studying of sdl1NahG and sdl1npr1-1double mutants.cDNA sequencing and RT-PCR showed the SDL1mutation in sdl1that changed splicing recognition sequence did not alter its splice site and the transcripts of the mutation SDL1gene in the sdl1mutant decreased markedly. By constructing the expression vector containing the endogenous promoter and35S promoter, we further detect the expression at transcriptional and translational levels. The expression at transcriptional level indicates that mutation at splicing site of sdl1doesn’t affect splicing efficiency. The gene is feedback controlled by its own promoter. In addition, the expression at transcriptional and translational levels of SDL1is not light-regulated.SDL1is an unreported new gene, bioinformatics analysis show that SDL1is highly conserved and contains important domains of fumarylacetoacetate hydrolase, suggesting its function as FAH functioning in tyrosine metabolic process. Learning from Tyr metabolism in vivo model, we treat plants with exogenous aromatic amino acids and their metabolic intermediates and end products to further study gene working site. The results show that exogenous high concentrations of Tyr and Trp can inhibit sdl1phenotype under short-day conditions, while there is no effect by adding exogenous Phe. The tyrosine content in sdl1mutant is not changed, illustrating a certain amount of Tyr and Trp seems to remedy the defect. Low concentration of metabolic intermediates homogentisate (HGA) in medium accelerates lesion necrosis phenotype of the sdl1mutants, while high concentrations of HGA in medium strongly inhibit plant growth. Metabolic end product fumaric acid (FA) has no impact to the phenotype of the plants, but the mutation of SDL1gene leads to reduction of FA content in the plants. The results indicate SDL1gene is likely to be working downstream of HGA, upstream of FA. But the phenotype is not due to the HGA accumulation and FA reduction.In summary, a short day-dependent lesion mimic SDL1gene is identified in this study, and its function is preliminary investigated. The findings are of great scientific significance to explore how the light length regulates the plant disease resistance.更多还原