A Study On The Application Of A PEG-mediated Transient Gene Expression System In Maize Mesophyll Protoplasts

Protoplast transient gene expression system has been applied to investigate plant signaling transduction pathways,screening functional genes of the signaling pathways and characterization of gene functions with the economic,simple,rapidly and efficient unique advantages,has become a powerful tool for studying plant physiological,biochemical and molecular mechanisms.Plant protoplasts display physiological properties and responses to hormones,metabolities,environmental cues,and pathogen-derived elicitors,similar to cell-autonomous responses in intact tissues and plants.The applications of protoplast transient transformation system have greatly contributed to high-throughput screening and systematic characterization of gene functions in combination with genetic,genomic,and transgenic approaches.At present,protoplasts have been extensively applied in dicot plants,such as Arabidopsis,tobacco and tomato,especially in Arabidopsis,the study of the PEG-mediated transient gene expression system using Arabidopsis mesophyll protoplasts has achieved a maturity stage,Whether in the study of promoters and signal transduction or in high throughput screening artificial microRNA(amiRNA)and functional genes,or the investigation of other aspects have made many important achievements.However,this system are far less studied in monocot plants such as maize and rice.Maize is not only an important crop plant,as well as a model monocot in plant research.Electroporation mediate transient gene expression system in maize protoplasts has achieved a lot of results for studying signaling transduction,promoter and the key enzyme regulate photosynthesis.Although chemical transfection method is easier to operate,but with PEG-calcium mediated transient transformation in maize protoplasts has rarely been reported.Owing to the maize transgenic engineering exist a lot of problems such as long-cycle needed,time consuming,consumables,big-workload and difficult to get the stable transgenic seedling,it is difficult to screening functional genes such as transcription factors and promoter and characterization of gene functions.While,the protoplasts transient gene expression system can rapidly,and efficiently screening the functional genes and analysis the functional characteristics.Therefore,transient expression techniques in maize protoplasts are necessary for gene function analysis.we used maize protoplast as the material,described in detail the methods of isolation,transfection and culture in maize protoplast.Choose the ZmMYC2-2 gene of maize,take PEG-calcium mediated the transfection,subcellular localize the ZmMYC2-2 gene,and did a further investigation of ZmMYC2-2 gene attranscription and secondary metabolities level.Otherwise,we identified the high-throughput screening system is also works in maize protoplast.After research and analysis,we got the following conclusions:(1)Subcellular localization assays show ZmMYC2-2 just expressed in nucleus.So presumably ZmMYC2-2 gene is similar to ATMYC2 gene of Arabidopsis thaliana,as a transcription factors potentially play an important role in regulating the synthesis and signaling transduction of Jasmonic acid(JA).(2)The results of overexpress ZmMYC2-2 gene suggest the expression of OPR7 and OPR8 genes related to JA synthesis were significantly upregulated,the expression of BXs genes related to benzoxaziniods synthesis were also significantly upregulated,and the production of benzoxaziniods were increased significantly.All of these results are coincided with the function prediction of subcellular localization results.(3)The results of overexpress ami RNA of ZmMYC2-1 and ZmMYC2-2 indicate the expression of both of them are significantly downregulated.It means these amiRNA that we selected target to the target genes respectively with a significant silencing.Above results demonstrate the PEG-mediated the transient gene expression system in maize protoplast is also as an important and practical tools for screening functional genes and characterization of gene functions.The point is this system provides an economic,rapid,simple and efficient method for the maize study at transcript and secondary metabolites levels.Meanwhile,it also provides a reference for maize transgenic work.