【作者】 李霞；

【导师】 董发才； 王二涛；

【作者基本信息】 河南大学 ， 生物化学与分子生物学， 2015， 硕士

【摘要】 80-90%的陆生植物包括重要作物水稻、小麦、玉米和大豆等都能与丛枝菌根真菌形成共生。在植物-丛枝菌根共生中,丛枝菌根真菌能够富集土壤中的磷、氮等营养,传递给植物供其生长;同时植物提供碳源供丛枝菌根真菌生长。植物与丛枝菌根真菌之间的信号交换是该共生关系建立所必需的。植物向根际周围释放信号分子如独脚金内酯,该信号被土壤中的菌根真菌感知,引起丛枝菌根真菌孢子萌发、分枝并分泌菌根因子(Myc factors)。菌根因子被植物的细胞膜表面受体所识别,通过一系列信号转导激活植物根部细胞核内的钙信号。钙信号可以被细胞核内一种钙调素依赖的蛋白激酶CCaMK/DMI3解析导致其自磷酸化,活化的DMI3磷酸化其底物IPD3,在转录因子的参与下,引起丛枝菌根共生相关基因的表达,进而建立共生。植物与丛枝菌根真菌共生的机制比较复杂,目前还有很多问题有待深入研究,比如:菌根因子的受体是什么基因以及其如何激活下游信号;共生信号通路中的GRAS类的转录因子如何激活菌根共生相关基因的表达等。本论文主要研究菌根共生相关转录因子对菌根共生基因的转录调控的分子机制。本实验主要是通过获取蒺藜苜蓿中一个GRAS类的转录因子MtDIP1的Tnt1插入突变体,来研究MtDIP1在菌根共生信号途径中的作用机制。研究发现DIP1的转录水平受菌根的诱导,dip1突变体影响菌根真菌的侵染,进一步的研究发现丛枝菌根标志基因MtLec5,MtGlp1,MtHA1,MtPT4和MtNR在dip1-1突变体中不能被正常诱导,说明DIP1参与了植物-丛枝菌根共生的信号转导。通过酵母双杂,pull dwon试验,我们进一步发现MtDIP1可以与菌根信号通路中调控菌根共生的关键基因DELLA,RAM1直接相互作用。体外转录激活实验表明DELLA-MtDIP1-RAM1通过形成复合体激活下游菌根共生基因的表达。本研究结果表明MtDIP1可能和菌根共生关键基因形成DELLA-MtDIP1-RAM1蛋白复合体调控植物-丛枝菌根共生。更多还原

【Abstract】 80%-90% terrestrial plants, including most crop rice(Oryza sativa), wheat(Triticum spp.), maize(Zea mays) and soybean(Glycine-max), are able to form symbiotic associations with arbuscular mycorrhizal fungi(AMF). The arbuscular mycorrhizal association that helps the host plant to uptake nutrients from the soil and is especially important for plant acquisition of phosphate and nitrogen, in turn the host plant provides carbonhydrate to the AM fungi.The early chemical communication between the host plant root and arbuscular mycorrhizal fungi(AMF) in the rhizosphere is essential for the iniation of symbiosis. Plant root releases signal molecules to rhizosphere such as Strigolactones which is perceived by AM fungi in soil and induces germination of AM fungal spores, hyphae branching and secretion of mycorrhizal factors(Myc factors). Myc factors are recognized by the plasma membrane receptor and active the calcium spiking in the nucleus of the cell. The calcium signal can be decoded by the calcium and calmodulin dependent kinase CCaMK/DMI3, which cause CCaMK/DMI3 autophosphorylation and phosphorylates IPD3 which is substrate of CCaMK. The mechanisms of symbiotic signaling between plant and AM fungi are still complicated and there are many questions need to be answered, Such as: What is the receptor of myc factors and how it actives the signaling pathway; What is mechanism underling the transcription factors active the expression of AM related genes and so on. Here we show the mechanism of transcription factors regulating the expression of AM genes during mycorrhizal symbiosis.In our study, we analysed the role of MtDIP1 in the AM symbiosis signaling pathway using two Tnt1 insertion mutants of GRAS transcription factor MtDIP1. Our results show that expression of MtDIP1 is up-regulated during mycorrhizal infection. The transcriptional level of DIP1 in dip1 mutants is great reduced and AM fungal colonization is also much lower than wild type. Expression of mycorrhizal marker genes Mt Lec5, MtGlp1, MtHA1, Mt PT4 and MtNR are not fully induced in dip1-1 mutant compared with wild type, indicating the function of MtDIP1 in AM symbioitic pathway. Using yeast two hybrid and pull down assay, we show that MtDIP1 physical interacts with MtDELLAs and MtRAM1 respectively, which both are required for mycorrhizal symbiosis. A transient reporter assay shows that MtDIP1 combined with DELLA and RAM1 actives the expression of AM symbiosis gene. We conclude that DELLA-MtDIP1-RAM1 may form protein complex regulates AM symbiosis through directly activating expression of AM symbioitic genes during mycorrhizal symbiosis.更多还原