Analysis Of Modification In Emf2B-jmj706Double Mutants And Flower Development Genes In Rice

Epigenetics refers to the case of no change in the DNA sequence of the gene, the function of gene has genetic changed, eventually leading to the change of phenotype. Now, epigenetic represents a new frontier of life science research. Histone modification plays an important role in epigenetic regulation. Histone modification is methylation, acetylation, phosphorylation, ubiquitination and other modify occurred on amino acid residues of histone. Types of the modified amino acid, location and types of modification constitute histone code, thus affecting chromatin structure and gene expression.OsEMF2b encodes H3K27methylation enzymes in the region of heterochromatin. The mutants which loss functions express as H3K27hypomethylation. JMJ706encodes H3K9demethylase in the region of heterochromatin. The mutants which loss functions performance H3K9hypermethylation. H3K9and H3K27methylation have related with methylation and transcriptional suppression. The homologous mutants which T-DN A inserts OsEMF2b have no capability of endosperm development, showing a pleiotropism phenotype:flowering time is advanced under long-day; development of some flowers is stopped prematurely; most of panicles can not stretch to the leaf sheath; development of floral organ is abnormal. Part of top spikelets of homologous mutants which T-DN A inserts JMJ706are developmental degradation, accompanied by the change of flower organ morphology and number. Loss of function of these two genes have led panicle developmental disorder, whether they have the same point in regulatory pathways, or regulate the same protein. We constructed double mutants by cross of emf2b and jmj706in order to study both regulation modes, whether the genes regulating methylation and demethylation exist interaction mechanism at the molecular level.Double mutants showed a fused phenotype of two mutants in plant types and floral organ in some degree, but the phenotypes were more closer to emf2b. We speculated that the regulations of histone modification between OsEMF2b and JMJ706may play a role by interaction. To reveal the mechanism, scanning electron microscope observation, dissecting microscope observation, identifying pollen sterility, RT-PCR, gene chip analysis and real-time quantitative PCR were used in research the double mutants. The main results are as follows:1. According to our statistics, heterozygous of emf2b and homologous mutants of jmj706were reciprocal cross, nine genotypes plants were isolated from F2. Although the actual proportion is not fully comply with the theoretical ratio, but the general trend is coincidence. Using RT-PCR, we respectively detected to transcripts(mRNA) of OsEMF2b and JMJ706in both wild-type and heterozygous mutant plants, but didn’t find in both of homologous mutants. Indicating that the mutant phenotypes of flower abnormalities were caused by insertional mutagenesis inactivation of OsEMF2b and JMJ706, emf2b and jmj’706mutants used in this experiment were stable materials.2. In the microarray analysis data of seedling stage and heading stage, we found that rice flower organ-specific gene OsMADS2,OsMADS6and OsMADS16expression levels did not change in seedling stage, but these three genes expression levels changed greatly in heading stage, decreased about44,53,18times respectively. Real-time quantitative PCR results showed that three genes’ expression levels of homologous mutants decreased28,55,25times in heading stage. Changes in three genes’ expression were the same trend by two methods, indicating that the microarray data was credible.3. We detected gene expression of double mutants by real-time quantitative PCR. Results showed that when the young panicles were3mm length, expression levels of three floral organ-specific genes were decreased in different samples. The decline multiples were emf2b>double mutants>jmj706, the decline multiple of double mutants was closed to jmj706. When pulvinus distance of young panicles was0, the trends of three floral organ-specific genes’ expression levels were difference in different samples. Changes of expression levels as follows:the decreased multiple of double mutants was less than the decreased multiple of emf2b, but the trend of jmj706was rising. As we observed, the double mutants fused the two parental traits, recovered the traits of emf2b and jmj706in some degree. Through reciprocal, we constructed double mutants and the phenotypes were similar. We speculated that OsEMF2b and JMJ706may have played a certain role in the double mutants, maybe there is a certain interaction between the histone modifying regulatory pathway of OsEMF2b and JMJ706.In summary, this research preliminary forecasts that OsEMF2b and JMJ706may exercise certain functions in the double mutants,OsEMF2b and JMJ706may regulate the way of histone modification by interactions. We will use chromatin immunoprecipitation to study proteins and their interactions in double mutants, find DNA fragments of specific binding by detecting target proteins. By proteins interactions and characteristics of DNA specific binding, we will deeply research specific ways that OsEMF2b and JMJ706regulate histone modification.