【作者】 叶敏；

【导师】 曹树青； 亓文明；

【作者基本信息】 合肥工业大学 ， 生物与医药（专业学位）， 2024， 硕士

【摘要】 镉(Cd)是一种对动植物有害的有毒重金属。随着人口增加及工业化活动的加剧,导致重金属Cd释放到环境中,使得土壤Cd污染问题日益加重。Cd在土壤中难以被降解,极易被植物吸收,使其通过食物链传递到人体内,从而对人类健康造成伤害。植物修复基因工程是目前解决土壤重金属污染的重要途径之一。然而,寻找和发掘耐受重金属毒害的关键基因并阐明其作用机理,是植物修复基因工程获得成功的关键。实验室前期研究已发现MYB4-MAN3-Mannose-MNB1信号途径通过GSH依赖的PC合成途径来调控植物Cd耐受。然而,对于MNB1结合甘露糖后调控的下游基因还未找到,因此本研究系统性地探究了Cd胁迫下该信号级联中的植物凝集素蛋白MNB1在结合甘露糖后其下游的作用机制。主要研究结果如下:(1)以MNB1为诱饵,利用蛋白质谱筛选鉴定发现RBOHD与MNB1互作,并用双分子荧光互补、免疫共沉淀和Pull-down方法进一步证实MNB1蛋白和RBOHD蛋白间确实有相互作用。(2)对野生型(WT)、rbohd突变体和RBOHD过表达植株进行Cd表型分析发现rbohd突变体植株与WT相比表现出对Cd敏感的表型,而RBOHD过表达植株表现出对Cd耐受的表型,表明RBOHD参与植物Cd胁迫响应的调节。(3)对WT植株和ProRBOHD:GUS进行Cd诱导处理并利用qRT-PCR和GUS染色法对其体内RBOHD基因的转录水平进行检测,发现RBOHD能够被Cd胁迫诱导表达。(4)为探究Cd胁迫下,RBOHD的可能作用途径,对WT、rbohd突变体和RBOHD过表达植株进行Cd胁迫处理,并利用原子吸收光谱法对其根部和叶部的Cd含量进行测定,发现与WT相比,rbohd突变体根部和叶部中的Cd含量明显降低,而RBOHD过表达植株根部和叶部中的Cd含量明显增加;进一步,通过对Cd胁迫下WT和RBOHD过表达植株外源添加丁硫氨酸亚砜胺(BSO)进行表型分析,发现BSO能够阻断RBOHD过表达植株Cd胁迫下的耐受表型,并且利用qRT-PCR检测Cd胁迫下WT、rbohd突变体和RBOHD过表达植株体内Cd胁迫响应相关基因表达,结果表明,与WT相比,rbohd突变体中PC合成途径关键基因PCS1、PCS2、GHS1、GSH2的转录水平显著降低,但RBOHD过表达植株体中则显著上升,表明RBOHD基因通过GSH依赖的PC合成途径来调控植物对Cd胁迫的耐受性。(5)进一步对RBOHD和MNB1基因的遗传关系进行分析。构建了35S:RBOHD/mnb1和rbohd/mnb1双突变体,并对其进行Cd胁迫表型分析,发现MNB1作用于RBOHD的上游来调控Cd耐受。RBOHD是植物体内重要的NADPH氧化酶基因,参与ROS的合成,为此,利用DAB染色法对Cd胁迫下WT、35S:RBOHD/mnb1、rbohd/mnb1双突材料体内的ROS信号水平进行检测,发现Cd胁迫下35S:RBOHD/mnb1体内的ROS信号强于WT,而rbohd/mnb1弱于WT,表明MNB1通过RBOHD介导的ROS信号来调控Cd胁迫响应。(6)为探究植物凝集素蛋白MNB1对RBOHD生化功能的影响,利用Cell-free体外降解实验分析,发现MNB1能够增强RBOHD蛋白的稳定性;进一步,通过对Cd胁迫下WT、rbohd、mnb1和rbohd/mnb1双突材料外源添加甘露糖进行表型实验,发现除WT表型增强外,rbohd、mnb1和rbohd/mnb1双突变体均不受其影响,表明Cd胁迫下Mannose调控MNB1-RBOHD介导的Cd耐受。总之,本文鉴定了一个新的调控植物Cd胁迫的关键基因RBOHD,并且其通过与Cd胁迫响应关键基因MNB1互作从而在植物抗Cd机制中发挥重要作用。本研究不仅揭示了一个新的调控植物Cd耐受的分子机制:Mannose-MNB1-RBOHD,而且为植物基因工程技术修复土壤重金属污染提供了新的基因资源和技术途径。更多还原

【Abstract】 Cadmium(Cd)is a toxic heavy metal that is harmful to plants and animals.With the increase in population and the intensification of industrialization activities,it leads to the release of heavy metal Cd into the environment,which makes the problem of soil Cd contamination worsen.Cd is difficult to degraded in soil,and it is very easy to be absorbed by plants,so that it can be passed to human body through the food chain,which can cause harm to human health.Phytoremediation genetic engineering is one of the important ways to solve the soil heavy metal pollution at present.However,finding and discovering key genes that are tolerant to heavy metal toxicity and elucidating their mechanism of action is the key to the success of phytoremediation genetic engineering.Previous studies in the laboratory have identified the MYB4-MAN3-Mannose-MNB1signaling pathway to regulate plant Cd tolerance through the GSH-dependent PC synthesis pathway.However,the downstream genes regulated by MNB1 after binding mannose have not been found,so this study systematically explored the mechanism of action of MNB1,a plant lectin protein in this signaling cascade,downstream of MNB1after binding mannose under Cd stress.The main findings are as follows:(1)Using MNB1 as bait,protein profiling screening was used to identify that RBOHD interacted with MNB1,and bimolecular fluorescence complementation,immunoprecipitation and Pull-down methods were used to further confirm that there was indeed an interaction between the MNB1 protein and the RBOHD protein.(2)Cd phenotyping of wild-type(WT),rbohd mutant and RBOHD overexpression plants revealed that rbohd mutant plants exhibited Cd-sensitive phenotypes compared with WT,while RBOHD overexpression plants were Cd-tolerant,suggesting that RBOHD is involved in the regulation of the Cd stress response in plants.(3)WT plants and ProRBOHD:GUS were subjected to Cd-induced treatment and the transcript levels of the RBOHD gene were examined in vivo using qRT-PCR and GUS staining,and it was found that RBOHD was able to be induced to express by Cd stress.(4)To investigate the possible pathways of action of RBOHD under Cd stress,WT,rbohd mutant and RBOHD overexpression plants were subjected to Cd stress treatment and the Cd content in their roots and leaves was determined using atomic absorption spectrometry,and it was found that compared with WT,the Cd content in the roots and leaves of the rbohd mutant was significantly reduced,whereas that of the roots and leaves of the RBOHD overexpression plants was significantly reduced.The Cd content in the leaves of RBOHD overexpression plants was significantly increased.Further,by phenotyping WT and RBOHD overexpression plants under Cd stress with exogenously added buthionine sulfoximine(BSO),it was found that BSO was able to block the phenotype of tolerance under Cd stress in RBOHD overexpression plants,and the Cd stress responses of WT,the rbohd mutant,and RBOHD overexpression plants in vivo were examined by qRT-PCR under Cd stress.The results showed that the transcript levels of key PC synthesis pathway genes PCS1,PCS2,GHS1,and GSH2 were significantly reduced in the rbohd mutant compared to WT,but increased in the RBOHD overexpression plants,indicating that the RBOHD genes regulate the Cd stress response of plants through the GSH-dependent PC synthesis pathway,and that the RBOHD genes regulate the plant’s Cd tolerance via the GSH-dependent PC synthesis pathway.(5)The genetic relationship between RBOHD and MNB1 genes was further analyzed.The 35S:RBOHD/mnb1 and rbohd/mnb1 double mutants were constructed and phenotyped for Cd stress,and it was found that MNB1 acted upstream of RBOHD to regulate Cd tolerance.RBOHD is an important NADPH oxidase gene in plants,which is involved in the synthesis of ROS.For this reason,the levels of ROS signaling were examined using DAB staining in the Cd stress in WT,35S.RBOHD/mnb1,and rbohd/mnb1 double mutant materials in vivo were examined using DAB staining,and it was found that the ROS signal in 35S:RBOHD/mnb1 was stronger than that in WT,while rbohd/mnb1 was weaker than that in WT under Cd stress,which indicated that MNB1 regulates the Cd stress response through RBOHD-mediated ROS signaling.(6)To investigate the effect of the plant lectin protein MNB1 on the biochemical function of RBOHD,the Cell-free in vitro degradation assay was utilized to analyze the effect of MNB1,and it was found that MNB1 was able to enhance the stability of the RBOHD protein;further,through the phenotyping experiments of exogenously added mannose to WT,rbohd,mnb1,and rbohd/mnb1 bipartite materials under Cd stress,it was found that be unaffected by the rbohd,mnb1 and rbohd/mnb1 double mutants,except for the enhanced WT phenotype,suggesting that Mannose regulates MNB1-RBOHD mediated Cd tolerance under Cd stressIn conclusion,this paper identifies a new key gene RBOHD that regulates Cd stress in plants,and it plays an important role in the mechanism of plant Cd resistance by interacting with MNB1,a key gene for Cd stress response.This study not only reveals a new molecular mechanism for regulating Cd tolerance in plants:Mannose-MNB1-RBOHD,but also provides new genetic resources and technological pathways for the remediation of soil heavy metal pollution by plant genetic engineering technology.更多还原