Functional Analysis Of PcG Gene OsEMF2b In Rice, Screening For Rice T-DNA Inserted Mutant Library To Get Reproductive Development Related Mutants

Rice is one of the most important food crops in the world, and also is a monocot model plant for studying the genetic development. Polycomb group protein (PcG) genes are a class of transcriptional repressor which is evolutionarily conserved among plants and animals. Several PcG protein complex in Arabidopsis have great important role in the control of flowering time and seed development. However, PcG genes function in rice is unclear. In this study, OsEMF2b, the PcG like gene isolated from rice was investigated by reverse genetics strategy with the T-DNA insertion mutants. The main results are as follows:1. Through bioinformatics analysis, we found two EMF2 genes OsEMF2a and OsEMF2b in rice, encoding a typical C2H2 zinc finger domain and a VEF-box domain. Alignment analysis suggested they are homologues of the Drosophila Su(z)12, Arabidopsis FIS2, VRN2 and EMF2. Evolutionary analysis show that EMF2 is an ancient and conserved gene, OsEMF2b had a most closely evolutionary relationship with sorghum EMF2.2. Real-time fluorescence quantitative PCR (qRT-PCR) analysis indicated that OsEMF2b genes tissues had a constitutive expression with a clear upward trend in the rice endosperm after fertilization, suggesting OsEMF2b involve in various periods of rice development.3. The result of cosegregation analysis of OsEMF2b T-DNA insertion mutants demonstrated that the mutant phenotype was caused by the T-DNA insertion. There was no obvious morphological difference between heterozygous mutant and wild type in vegetative growth stage. Besides, abnormal shape of flower organs and the number of variation during reproductive growth stages were observed; vegetative growth of homozygous mutant was significantly inhibited, and in the last, the plant did not flower under long-day conditions, revealing that rice OsEMF2b genes involved in regulation of vegetative growth, floral organ development and flower transformation.4. RT-PCR analysis showed some MADS-box genes were differencialy expressed in wild-type and mutant during panicle development, suggested OsEMF2b gene may affect floral organ development by regulate of MADS-box genes.5. Osemf2b homozygous mutants did not flower under long-day conditions, indicating that the OsEMF2b gene promote flowering under long-day conditions in rice. Further analysis of the diurnal expression patterns displayed that, OsEMF2b gene had the highest expression during the dawn of the day in short-day condition and the lowest at noon. The expression of some known Genes, promoting flowering in rice, such as OsMADS50, Ehd1, Hd3a, OsMADS14 and OsMADS15 in Osemf2b homozygous mutant were significantly reduced, pointing out that OsEMF2b genes involved in rice photoperiodic flowering pathway mediated by Ehd1, and induced flowering by promoting expression of the FT-like gene Hd3a.6. Construction of over-expression vector and complementarity vector of OsEMF2b gene was fulfilled, which may set up a foundation further analysis of the phenotype of transgenic plants to determine the function of this gene.In recent years, the establishment of several major world rice T-DNA insertion mutant library and the rice genome sequencing data provides an important resource for people to study function of the rice genome by using forward genetics and reverse genetics strategy. In this paper, targeted on the rice flower organ and seed development defects related phenotype, a large constructed rice T-DNA insertion mutant library, constructed by National Plant Gene Research Center of Huazhong Agricultural University, was screened and obtained a large number of mutant information and materials for further cloning floral organ development in rice and seed development related genes. Detail results are as follows:1. In the 9760 stable lines of phenotype variant,177 lines show morphology and number change of floral organ, with mutation frequency of 1.81%; 179 lines of the 3432 lines show seed development defect, the percent of mutants was 5.22%.2. The 270 interested lines of phenotype variant are obtained and 175 lines show T-DNA insertion through PCR testing, the positive rate is 64.8%.3. Use flanking sequence given by rice T-DNA insertion mutants public databases (RMD), We identified a low setting rate percentage mutation, its mutant morphology was confirmed to be cosegregated with their corresponding T-DNA flanking sequences tags, providing an excellent genetic resource for further studying of this gene function.