Isolation Of Maize Endosperm Protoplast And Establishment Of Instantaneous Transfection System

Maize is one of the most important food and feed crops in the world,and it has become the food crop with the largest planting area and total yield in China,so improving the yield and quality of corn is of great significance to the food security in the new normal period.70-90%of the dry matter in corn grains is stored in the endosperm,which is not only the main storage organ of corn,but also the key factor to determine the formation of corn economic yield.The development,proliferation and enrichment of endosperm cells determine the yield and quality of maize grains,so in-depth study of the development of maize endosperm cells is of great significance to improve the yield and quality of maize.At present,stable transformation is mainly used in the study of genes related to endosperm traits in maize,but it has the disadvantages of long period,complex operation and poor stability,while instantaneous transformation is fast,convenient and efficient.Plant transient transformation system mainly uses onion epidermal cells,suspension cells,calli,tobacco leaves and protoplasts.Because there is no limitation of cell wall,protoplasts can absorb foreign macromolecules relatively easily,so protoplasts have become a general cell experimental system for basic theoretical research and bioengineering technology.The protoplast transient transformation system enables the rapid expression of foreign genes in cells,and studies the behavior of genes in cells by detecting the expression of target genes.It has been widely used in gene expression regulation,promoter analysis,chromosome immunoprecipitation,protein subcellular localization,protein interaction,protein activity detection and signal pathway.At present,there is no report on the instantaneous transformation system of Gramineae endosperm protoplasts,so in this study,protocols for isolation and transfection maize leaf mesophyll was followed with little modificatons.Using response surface methods,the optimum conditions for the protoplast isolation and PEG-Ca²⁺-mediatedtransfection of maize endosperm tissues were established.The established endosperm protoplast system was applied to some cellular and molecular biology experiments.The main results are as follows:1.Optimization of isolation conditions of maize endosperm protoplasts.Corn ear at 6-10 days after pollination（DAP）was harvested,and the seed coat was carefully removed to excise theendosperm cells.The enzymatic hydrolysis solution was prepared for protoplast isolation.The isolation factors;cellulase,macerozyme and mannitol cooncentrations and hydrolysis time,were optimized by Box-Behnken design.The optimal and quality protoplast yield were obtained at 1.0%cellulase,0.75%macerozyme,0.4M mannitol,and 6h duration of hydrolysis.Under this optimal conditions,maize endosperm protoplast yield was about 2.43×10⁶/m L,with more than 95%of the cells remained in the suspension solution as confirmed by FDA staining.2.Optimization of the instantaneous transfection conditions of maize endosperm protoplasts.The transfection efficiency of the PEG-Ca²⁺-mediated transfection conditions:endosperm age,protoplast and plasmid concentrations,were optimized by Central Composite design.The results showed an optimal transfection efficiency of64.8%when 1.5×10⁶ cells/m L of protoplasts,10μg plasmid DNA and endosperm at 8DAP were used for transfection system.3.Applications of the established maize endosperm protoplast system in various molecular studies:（1）Subcellular localization detection:ADPG transporter（BT1）is a class of transmembrane protein,whose main function is to transport ADPG,the direct precursor of starch that is synthesized in the plastids of endosperm cells.We used the fusion expression vector,p CAMBIA2300-Zm BT1-GFP,as the experimental group and p CAMBIA2300-GFP vector as the control group.The vectors were transiently transfected into maize endosperm protoplasts,and subcellular localization was investigated under the laser confocal scanning microscope.The fluorescence signal of the control group was distributed in the entire endosperm protoplast cells,but the green fluorescence signal of the transfected system containing the Zm BT1,was observed in the plastid membrane of the protoplast cells.This confirmes the application of maize endosperm protoplasts in the subcellular localization of protein expression study.（2）Bimolecular fluorescence complementary（Bi FC）analysis:Maize endosperm specific transcription factor O2（Opaque 2）and PBF1（Prolamine-box binding factor）,regulate the nutritional quality of maize grain mainly by regulating the synthesis of corn zein gene.The recombinant vectors:GN-PBF1 and GC-O2 were used as the experimental group,and GN/GC-O2 and GC/GN-PBF1 as the control group.The constructed vector pairs were co-transfected into maize endosperm protoplasts.The interaction of the two transcription factors was confirmed under laser confocal scanning microscope.In the results,green fluorescence signal at the nucleus of the protoplast cells,was detected in transfection system of the experimental group,but no green fluorescence signal was detected in the control group.The results confirmed the nuclear interaction of GN-PBF1 and GC-O2 in maize endosperm protoplasts.This indicates that the maize endosperm protoplast system can be used for protein-protein interaction study.（3）Analysis of foreign protein by Western Blotting:Maize endosperm protoplasts were transiently transformed with p BI221-GFP as the experimental group and p BI221 empty vector as the control group.The total protein of transformed maize endosperm protoplasts were extracted and analyzed by Western Blot with GFP antibody.The results showed that p BI221-GFP was successfully expressed in maize endosperm protoplasts.This suggests that the maize endosperm protoplast system can be used to analyze the expression of foreign proteins.（4）Analysis of the regulatory pathway of gene expression:Using p BI221-Zm MYB14 as the experimental group and p BI221 empty vector as the control group,the maize endosperm protoplasts were transiently transfected with the vectors.RNA was extracted from the transfected maize endosperm protoplasts,and reverse transcription was performed.The expression of Zm MYB14 was analyzed by fluorescence quantitative PCR（q PCR）.At the same time,the effect of Zm MYB14 on p Zm BT1 promoter activity was analyzed.The results showed that Zm MYB14 was successfully overexpressed in transfected endosperm protoplasts,and the overexpression of Zm MYB14 promoted the activity of p Zm BT1 promoter,which was consistent with previous studies.This proves that the maize endosperm protoplast system is suitable for q PCR analysis.The above results show that the endosperm protoplast transient transfection system can be used as a model system for the study of protein subcellular localization,protein-protein interaction study by Bi FC,protein expression,q PCR transient gene expression and gene regulation pathway analysis.The system endosperm protoplast system will be a powerful tool for rapid analysis of a large number of genes related to endosperm traits,and will be of great significance for the study of improving grain yield and quality.