The Cloning And Functional Analysis Of Glucan Endo-?-1,3-glucosidase Gene ZmEGP In Maize

As an important crop for food,industry and fodder,the development of maize(Zea Mays L.)was closely related with food security.Studying maize?-1,3-glucanase,which participate in almost all of the plant growth and development stages,and have resistance to disease or stress,could provide some help to research in grain development and breeding associated with yield as well as the resistance.By homologous cloning we obtained two c DNA sequences and its corresponding genome sequences of ?-1,3-glucanase from two different maize inbred lines Dan232 and N04.A series of functional prediction and research assays were ultilized in this study,such as subcellular location prediction and domain analysis.We constructed an overexpression vector to transformed it into Arabidopsis thaliana and also designed a RNAi expression vector to maize.The results laid a good foundation for further study,such as protein purification,enzyme activity assay,and other functional research for Zm EGP.The main results were as follows:1.Our laboratory have built protein libraries for pericarp and endosperm at different grain filling stages from the dent corn inbred Dan232 and popcorn inbred N04.A peptide namely Zm EGP from the protein libraries were chosen to search its homologous gene in NCBI,We obtained its c DNA and genome sequences by homology cloning techniques.The full length c DNA was consisted of a 14 bp 5'untranslated region(UTR),a 232 bp 3'UTR,and a 1389 bp complete open reading frame(ORF).Comparing the two c DNA and DNA sequences of Zm EGP between the two inbreds,we found that the main differences were existed in the first intron except for the single nucleotide polymorphisms(SNP)in coding sequence.Using bioinformatics software to analyze the protein domains and physicochemical property,the results showed that Zm EGP was grouped to GH17 according to its GH17 and X8 domains.2.Phylogenetic analysis indicated that Zm EGP was closely related withmonocotyledons but not to dicotyledons.The Zm EGP amino acid sequence was much similar to sorghum bicolor and Setaria italica,with 90% and 86% amino acids identy.Through comparison with the gene's genome sequence with published B73 complete genome sequence in Maize GDB,we speculated that Zm EGP was most likely to be located on the second chromosome.3.Real time PCR(RT-PCR)analysis showed that Zm EGP was an tissue non-specific expression gene.The expression trend of the gene was mostly identical in maize seedlings,which manifested as stem > leaf > root,and the relative quantitative expression was higher in maize inbred line Dan232.In various maize grain tissues,Zm EGP expressed highly in 33 DAP endosperm in Dan232,but at other stages and tissues its expression was weaker for Dan232 than that for N04.4.The recombinant prokaryotic expression vector p EASY-Zm EGP was constructed.Fusion protein was induced with 0.5m M IPTG to expression and examined by SDS-polyacrylamide gel electrophoresis(SDS-PAGE).The result showed that there was an obvious expression increased band at 47.80 k Da.The western blot analysis was consistent with Prokaryotic expression.This result indicated that Zm EGP could be induced to expression in vitro,which provied a base for the protein purification and enzyme activity analysis.5.Western blot assay was ultilized to detect the distribution of Zm EGP protein in two maize inbred lines.The results suggested that the signal of the target protein existed both in pericarp and endosperm.The signal in pericarp was weaker than that in endosperm,especially in pericarp at 10 DAP.Combing these appearances with RT-PCR result,we found that the Zm EGP protein was mainly expressed in endosperm and expressed higher in N04 than in Dan232,which showed a good agreement with protein quantification result.6.Overexpression vector was constructed and transformed into Arabidopsis thaliana.The transgenetic plants were selected up to T2 by hygromycin resistance.The positive plants and negative control were treated by drought or high salt stress.The performances for the transgenetic plants were consistent with the negative control plants under drought stress,but they were different in high salt stress treatment.The transgenetic Arabidopsis thaliana plants showed high salt stress tolerance.