Cloning And Expression Of Four Polycomb Gene In Maize

Maize is one of the the major food and feed crop all over the world,has an important role in agricultural production.Carrying out research on development and maize-seed development especially of maize would be of great significance for maize variety improvement and grain production.Recently,epigenetic studies become the research frontier and hot spot based on genotype and phenotype correlation.Study of epigenetic of corn growth related genes,would explore the new train of thought for further maize yield increase.In this study,Ch7-2 variety of maize was choosed for the research material.N-terminal of the base sequences of important genes of Polycomb Group(PcG)protein family,including fie1,mezl,epc101 and vef101 were cloned into pMD19-T plasmid from leaf,stem tip,coleoptile and root of corn at different period before earing-stage and sequenced;each gene fragements were inserted into pGEX-6p-1 plasmid for prokaryotic expression,the Gene expression product were useed to immunize the animal for antibody preparation;Finally,Western blot tests were applied for estimating the binding effect of expressed protein and antibody prepared in this study.This research would provide the premise for fishing for combining with the related DNA sequence in subsequent study,and laid a solid foundation for deeply analyzing the role of PcG genes played in the development of maize seed pattern.The results were displayed as followed.1.PcG protein family related genes expressed more highly in 8 d in grain after pollination,lowly in 6 d and 18 d,nearly not in 4 d after pollination.2.After sequencing,fragment length of fie1,mez1,epc101 and vef101 was confirmed to be 357 bp,360 bp,306 bp and 405 bp,respectively.The gene identity between Ch7-2 and B73 variety logged-in GenBank was 98.9%、100%、100%and 99.2%in turn.3.PcG genes,fie1,mez1,epc101,vef101 were inserted into pGEX-6p-1 plasmid and sequencing for characterization,followed by detection of enzyme digestion and PCR test.Each expression vectors was trasfected into the competent cell of express strain BL21(DE3),respectively,and then undergone induction for 2 h or 4 h with a final concentration of 0.5 mM of IPTG.The results of soluble analysis showed that the obtained the recomninated PcG proteins were expressed in inclusion body form.4.To get antibodies specific for expressed protein,the harvested precipitation were subjected to sds-page,the gel with a single protein band was dissolved in the Tris solution after pounding.Centrifugal supernatant was using for animal immunization.5.The Western hybridization experiments showed that antibody preparation in this study could bind for fiel.