The Screen And The Biology Function Research Of Type 2 Metallothionein From Puccinellia Tenuiflora And Arabidopsis Thaliana Interaction Protein

Metallothioneins(MTs)are a family of low molecular mass,cysteine-rich and metal-binding proteins in plants,animals,fungi and cyanobacteria.Plants MTs functioned in metal detoxification and homeostasis in tissues,regulated the transport of metal ions,and cleared ROS,which endowed MTs the role that can enhance the plants tolerance to stresses.Current studies indicate that MTs protein are involved in many important physiological and biochemical processes,and MTs are an important class in plant defense system.However,the studies on its physiological function in plants are not very thorough,and some phenomena are unexplained.The expression of PutMT2,a type 2 metallothionein of Puccinellia tenuiflora,were induced by NaCl,H2O2,CuCl2 and other abiotic stresses,and can improve the transgenic yeast tolerance to high salt,metal ions such as ZnCl2,FeCl3,which indicate PutMT2 may involved in adversity adaptation.The interactions with other proteins are inseparable for various proteins exerting functions in cell.Obtaining the interacting protein of metallothionein and analysis of the protein functions would help explain the role of metallothionein in improving plant resistance and growth.In present study,in order to better understand the function and role mechanism of metallothionein in plant cells,the metal binding function of PutMT2 protein was studied,the interacting proteins of type 2 metallothionein were screened by yeast two-hybrid and their roles in oxidative stress responses of plants were analyzed.The main results are as follows:1.PutMT2 gene could express in roots,stems,leaves,flowers and sheath,between which the sheath was the highest,and the expression in leaves was higher than in roots;the expression of PutMT2 were induced by NaCl,H2O2,CuCl2,and ZnCl2 stress;PutMT2 protein localized in the cytoplasm.PutMT2 protein could bind with Zn ions and Na ions,then increased the transgenic yeast resistance to ZnC12 and NaCl stress;PutMT2 protein could bind with Cu ions but transgenic yeast became sensitive to CuCl2 stress.In these three ions the binding capacity of PutMT2 to Cu ion was strongest,to Na ion was weakest.2.The yeast two-hybrid were used to screen the interacting proteins of PutMT2 and two candidate target proteins were isolated,which were mitochondrial outer membrane voltage-dependent anion channel 3(AtVDAC3)and thioredoxin m2(AtTrx m2),and Arabidopsis type 2 metallothionein AtMT2a and AtMT2b also can interact with AtVDAC3 and AtTrx m2 in yeast level.Both AtMT2b and AtMT2a can interact with AtVDAC3 respectively were identified by yeast two-hybrid system,and this interaction was specific with AtVDAC3 for the negative interaction with other VDAC,and the interaction region was the N-terminal of At VDAC3;Pull Down experiment confirmed the direct interaction in vitro of AtMT2a and AtMT2b with AtVDAC3 respectively;and bimolecular fluorescence complementation experiments(BiFc)confirmed AtMT2a and AtMT2b can interact with AtVDAC3 respectively in vivo,and the interaction position was in the mitochondria.The GFP fluorescence localization analysis showed AtMT2a and AtMT2b are cytoplasmic proteins,and AtVDAC3 protein localized in mitochondria.3,metallothionein are thiol-group-rich proteins,and involved in intracellular ROS detoxification;as a mitochondrial outer membrane channel VDAC is closely related to the intracellular redox state.So we mainly studied the role of AtMT2b and AtVDAC3 in plant oxidative stress response to understand their functions in plants,then analyzed the interaction mechanism and significance of these proteins.At first we studied the gene expession pattern of AtMT2b and AtVDAC3 repectively in Arabidopsis by real-time quantitative PCR.The results showed that AtMT2b and At VDAC3 can expressed in all tested tisusses,with higher expression leavel in younge leaves than in mature leaves and senescent leaves,but AtMT2b gene expression in young leaves was higher than in roots,while At VDAC3 gene expression in roots was higher than in leaves;and the expression of two genes were induced by oxidative stress such as high salt,osmotic,metal ions,exhibiting relationship with oxidative stress response.Then we analyzed the growth phenotype and the ROS production of the mutant and transgenic Arabidopsis plants of AtMT2b and At VDAC3 under various oxidative stress respectively;and analyzed the influence of AtMT2b and At VDAC3 on the ROS production and mitochondrial membrane potential(MMP)under NaCl treatment.The results showed that:AtMT2b overexpression increased the Arabidopsis resistance to NaCl,H2O2,CuCl2,ZnCl2,CdC12 stresses and reduce ROS levels.and the mitochondrial membrane potential was maintained under NaCl stress,while AtMT2b mutation made the Arabidopsis more sensitive to oxidative stress and with more ROS accumulation,and lower mitochondrial membrane potential than WT under NaCl stress.AtVDAC3 mutant plant advdac3#7 showed stronger resistance and lower ROS levels under NaCl,H2O2,CuCl2,ZnCl2,CdCl2 stresses than WT,and the mitochondrial membrane potential was higher than WT under NaCl stress when the cells ROS was in lower levels,while overexpression of Arabidopsis AtVDAC3 showed the opposite phenotype.These results indicated that AtMT2b and AtVDAC3 could play a role in the Arabidopsis stress response,and could affect the ROS production and mitochondrial membrane potential state within the cells under salt stress.But the affection of AtMT2b to Arabidopsis stress response was opposite with the affection of AtVDAC3,so AtMT2b might negatively regulate the AtVDAC3 channel.Over all,the Arabidopsis type 2 metallothionein AtMT2a and AtMT2b interact with mitochondrial outer membrane voltage-dependent anion channel 3(AtVDAC3).AtMT2b and AtVDAC3 play a great role in plant denfense.But the interaction mechanism need furture studies.