Corn Genome Dna Methylation Level And Its Relationship With Heterosis,

DNA methylation is an important pattern of the epigenetic modification,generally existing in the genomes of the plant and the animal,and playing an important role in controlling the gene expression and maintaining genomic stability.Through the process of demethylation and methylation of the cytosine in the DNA sequences,plants growth and development are regulated. The way of breeding of maize changes from traditional breeding to molecular breeding,which is the main material for hybrid breeding.More and more studies indicate that maize endosperm development is controlled by the maternal genomic imprinting,whereas some genes from the paternal genome are methylated and kept silent.Elucidation of the mechanism of epigenetics during endosperm development will largely contribute to our endeavor to improve the quality or production.Methylation-sensitive amplified polymorphism(MASP) was firstly reported by L Z Xiong who studied the differences of the genomes methylation between the rice F1 and their parents, which was based on the AFLP method,based on two enzymes sensitive to the methylated cytosine. By using a little genomic DNA,the variation of methylation can be chedted.Up to now,it is prevalently adopted in many labs to study the different methylation levels.Here,we use corn species Zhengdan958 as material,which grows prevantly in china.We try to screen genes which involves in heterosis.Zheng58 is the female parent,Chang7-2 is the male parent.At 10-15 DAP(days after polination),endosperm cell mitosis stops and begin to accumulate nutrient material.Up to now,there are a lot of reports about the maize methylation at the early stage of the seed development,however,few report were reported about the later stage.28DAP seeds are choosed as material to study the methylation variation.Later,fragements which show demethylation are cloned and for futhur study.All of the results were summarized as the following:1.In this study,checking the production of pre-PCR and selective-PCR on the 1.2%(w/v) agarose gel,the bands of the pre-PCR show smear because their size were approximate to each other.The bands of the selective-PCR were assembled and around the 500bp,it was obvious that the methylation level varies among the materials.This pre-pcr amplication and selective-pcr selection greatly improve the efficiency.2.All the bands of the MSAP analyzed,and separated five types according to their transformation's trend:A type was the all-methylation,B type was demethylation,C type was hyper-methylation,D type was hypo-methylation,E type was up-methylation.Among them.the B type and C type occupy most of the bands,the ratio 33.7%and 35.8%respectively.3.In the study,we observed that the level of the unmethylated cytosine in the F1 generation is higher than its parents,whereas the level of the methylated cytosine is lower than its parents;which caused by the level of the demethylation simultaneously decreases in the F1 generation.After hybridization,the methylation pattern of F1 generation changed to harmonize the expression of genes from both parents.Some genes express with high level after demethylated,whereas,other genes are restricted by methylated,which may be affect the heterosis.4.By the F1 generation's pattern of the methylated inheritance,we found that 46%from maternal lines,howere,only 4.8%from paternal lines which indicated that the F1 inherit mostly came from maternal lines.Of the patterns of methylation,semi-methylation was dominate,whith a ratio of 28.9%of total MSAP bands.5.Eleven differentiated bands of F1 generation DNA demethylation were cloned and analyzed against the maize genome.These methylation sites alteration occurred both in promoter region and coding region.Among the cloned fragments,E4H6 encodes a protein homologous with the member of F-box-like family protein in rice,which is the componets of the SCF complex involving in ubiquitim proteosome pathway.E2H5-1,E2H5-3,E1H5-1,E3H4-1 and E3H4-2 encode some hypothetical protein or unknown protein;whereas,E2H5-2,E1H1-3 and E1H3-1 are expressed sequence tags.