【作者】 徐进；

【导师】 周艳虹；

【作者基本信息】 浙江大学 ， 蔬菜学， 2023， 博士

【摘要】 番茄(Solanum lycopersicum)是我国重要的蔬菜经济作物。然而,冬春季节频繁出现的低温冷害极大地限制了番茄植株的生长和果实成熟等生长发育过程,导致产量下降和品质变劣。因此,探究番茄低温抗性和果实成熟的调控机制,对于保障冬春季节番茄优质高产和可持续生产具有重要的意义。研究表明,O-糖基化修饰对维持植物的正常生长至关重要。本文以番茄为研究对象,探究了O-糖基化修饰在番茄逆境响应以及生长发育中的作用,并阐明了O-GlcNAc糖基转移酶SlSEC和O-岩藻糖基转移酶SlSPY在番茄抵御低温胁迫以及果实成熟过程中的调控机制。所得主要结果如下:1.鉴定了O-糖基转移酶SlSEC与SlSPY在不同生长发育时期以及在不同非生物胁迫条件下的表达水平以及突变体表型。SlSEC和SlSPY基因在不同组织各时期生长发育过程中的表达水平呈现动态的变化,且参与多种非生物胁迫的响应。此外,slsec1和slspy突变体表现出不同程度的缺陷,与野生型相比,slsec1突变体仅表现出轻微的生长缓慢,而slspy突变体表现出更为明显的生长缓慢和植株矮小。2.明确了SlSEC1通过提高低温负调控因子SlC3H39蛋白的稳定性,负调控番茄的低温抗性。通过酵母双杂交鉴定到SlSEC1与SlC3H39互作。SlC3H39基因在低温胁迫下表达上调,且SlSEC1通过对SlC3H39进行O-GlcNAc修饰提高了SlC3H39蛋白稳定性,进一步促进SlC3H39蛋白在低温下的积累。通过构建过表达等遗传材料,证明了SlC3H39负调控番茄低温抗性,且SlC3H39基因的突变部分恢复了SlSEC1过表达材料的低温抗性。3.解析了CCCH串联锌指蛋白SlC3H39通过介导m RNA降解负调控低温抗性的机制。通过转录组分析表明,slc3h39突变体中抗性相关基因呈现上调趋势而生长发育相关基因呈现下调趋势。体内瞬时表达以及体外核酸结合试验明确了SlC3H39具有RNA结合能力并参与介导RNA的降解。通过将转录组与RNA免疫共沉淀结果相关联,鉴定到SlC3H39蛋白结合的靶标基因,发现SlC3H39通过结合低温响应基因的3’UTR介导其降解,从而负调控番茄的低温抗性。4.明确了SlSPY正调控番茄果实成熟。通过检测果实成熟指标类胡萝卜素积累和果实软化程度,发现SlSPY基因能够促进番茄果实的成熟进程。然而,SlSPY基因不影响果实中乙烯的合成。利用外源乙烯处理后,发现SlSPY基因促进番茄果实对乙烯的响应,正调控果实成熟。5.解析了SlSPY-SlEIN2模块通过促进乙烯信号转导正调控果实成熟的机制。通过酵母双杂交鉴定到番茄中的O-岩藻糖基转移酶SlSPY与乙烯信号通路中的重要组分SlEIN2互作,通过检测SlSPY过表达材料中SlEIN2蛋白在不同细胞组分中的含量,发现SlSPY可以提高SlEIN2蛋白的稳定性,并促进其在细胞核内的积累,进而促进乙烯信号转导。综上,本文揭示了植物O-糖基转移酶SEC和SPY在逆境响应以及生长发育过程中的作用。鉴定了SlSEC1-SlC3H39通过转录后调控植物低温抗性的机制,并揭示了SlSPY-SlEIN2在果实成熟中的作用及机制,为番茄的生长发育调控以及高产优质生产提供理论支撑。更多还原

【Abstract】 Tomato(Solanum lycopersicum)is an important economic vegetable crop in China.However,the frequent occurrence of cold damage in winter and spring severely impairs plant growth and productivity.Plants undergo a range of changes to survive from cold stress.The physiological and biochemical methods to improve the cold resistance and shorten the growth cycle were used to minimizing damage and ensure quality of tomato.In this study,vegetable crop tomato was used to explore the role of O-glycosylation in defense responses and growth processes.The regulatory function of Oglycosyltransferase SlSEC and SlSPY in tomato cold tolerance and fruit ripening was identified.The main results are as follows:1.The transcript abundance of SlSEC1,SlSEC2 and SlSPY in different growth stages and during variant abiotic stresses response was identified.The transcript level of SlSEC and SlSPY in different tissues showed dynamic changes during growth and development.SlSEC and SlSPY were also involved in the response to various abiotic stresses.In addition,the slsec1 and slspy mutants exhibited different degrees of defects.Compared with the wild type,the slsec1 mutants only showed a slight semidwarf phenotype,while the slspy mutant showed more obvious slow-growing and had a dwarf phenotype.2.The O-GlcNAc transferase SlSEC1 was found to negatively regulates cold tolerance in tomato by promoting the stability of SlC3H39,which is a negative regulator of cold tolerance.The interaction between SlSEC1 and SlC3H39 was screened through yeast two-hybrid.Further study shows that SlSEC1 physically interacts with SlC3H39 proteins and promotes the stability of it by modulating O-GlcNAclation status of SlC3H39.SlC3H39 is rapidly induced by cold stress and negatively modulates plant cold tolerance.The slc3h39 mutants partially rescued the cold tolerance of SlSEC1 overexpressing lines.3.SlC3H39 negatively modulates plant cold tolerance through post-transcriptional regulation by binding to cold responding m RNAs 3’UTR and reducing those transcripts.The RNA-seq results shows that genes involved in defense response and cold response are up-regulated and genes involved in growth such as meristem development and photosynthesis are down-regulated in slc3h39 mutants compared with WT.Transient expression in vivo and nucleic acid binding assays in vitro confirmed that SlC3H39 had RNA binding ability and was involved in mediating RNA degradation.By associating the RNA-seq with RNA immunoprecipitation-seq,we identified that SlC3H39 binds to3’UTR of cold responding gene and decaying those transcripts.4.SlSPY positively regulates fruit ripening by changing the ethylene response in tomato.The accumulation of carotenoid content and fruit firmness in the WT,slspy mutants and SlSPY overexpressing lines indicate the positive role of SlSPY in fruit ripening.Further study indicates that SlSPY-regulated fruit ripening is closely related to ethylene response in tomatoes.5.SlEIN2 is essential for SlSPY in regulating fruit ripening in tomatoes.A central regulator of ethylene signaling SlEIN2 is a SlSPY interacting protein.Mass spectrometry analysis further identified that SlEIN2 has potential sites of O-glycans modifications.SlSPY promotes the stability and nuclear accumulation of SlEIN2,thus positive regulates the ethylene-promoted fruit ripening in tomato.In conclusion,this study reveals the role of plant O-glycosyltransferases SEC and SPY in defense responses and growth processes.we propose a SlSEC1-SlC3H39 module,which modulates tomato cold tolerance through post-transcriptional regulation.And a novel insight into the role of the SlSPY-SlEIN2 module in tomato fruit ripening was proposed.These findings provide positive guides for regulating the development and formatting the yield and quality of tomato.更多还原