The Initial Function Analysis Of The Downstream Genes Of EFD In Arabidopsis

Epigenetic regulation refers to the changes of gene expression in the case of no nucleotide alters,DNA methylation can change the activity of DNA without any alter in DNA sequece.In plants,DNA methylation happens in three different context,including CG,CHG and CHH.The bisulfite sequencing method is the most accurate method for the detection of methylation,the methylation level is detected in the genome exactly to every single cytosine nucleotide,which is called the "gold standard".Microsporogenesis is ciritical for plants to produce normal male germ cells.The early finding of our laboratory is gene EFD,which has a relationship with the anther development in early stage.Through analysis of the mutant plant,EFD gene is involved in the regulation of primexine and callose formation in the early stage of microspore development,indicating that this gene plays an important role in the process of Arabidopsis pollen wall patterning.Bioinformatics analysis of EFD protein shows it has a typical methyltransferase structure,and a specific domain of DNA methyltransferase as the substrate of DNA.Compared to the several known plant methytransferases,we find EFD has a closest genetic relationship with Dnmt3 family methyltransferases which show a de novo methyltransferase activity.In vitro enzyme activity experiments confirms that EFD has a methyltransferase activity.But the mechanism of EFD regulation is not very clear now,this paper aims to find out mechanisms of downstream genes of EFD protein,and reveal how EFD protein regulate downstream genes' expression to control the process of anther development.Higher plant sexual reproduction process not only is the main way to plant reproduction,also is foundation of human survival food.As the male reproductive system of sexual reproduction.The normal development of anther and pollen has great significance to the process of sexual reproduction.For this purpose,we carried out the following experiment to find the downstream genes:1.Our purpose is to find the downstream genes of EFD protein,methylation can inhibit gene expression,so our interests turn to genes whose expression levels has a up or down transform in efd mutant background compared to col.According to Hu's microarray data,we identified a significant up-regulated gene in the efd mutant background as a downstream candidate,and named it ER3.By extraction of inflorescence RNA,we performed real-time PCR experiment,confirming the up-regulated situation of ER3 in the efd mutant background.RT-PCR and GUS staining results showed that ER3 gene is a universal expression gene.2.We purchased the single mutant of ER3 gene from SALK seed company and indentify the homozygous lines.RT-PCR of ER3 gene in the selected mutant showed little expression,indicating it's a valid mutant.Then we performed hybridization,efd mutant as female parent and er3 mutant as male parent.Through genome screening,we confirmed the heterozygosis F1 generation.After F1 generation selfing,we select double homozygous mutant in the F2 generation.3.The efd mutant shows a pollen abortion phenotype,early siliques' development is not normal,but in the end it will restore fertility.So we counted the number of ovules in the efd/er3 double homozygous mutant.We found that siliques of the double mutant recovered earlier than efd mutant and formed masses of fertility ovule.Alexander staining of anther and pollen germination in vitro experiment showed the pollen vigour of efd/er3 double is good.4.To further determine whether ER3 gene is a direct target gene of EFD protien,we examed the methylation level of ER3 gene in promoter region.The result indicates that the promoter region of ER3 in efd mutant background and the wild type don't show any significant difference.The expression of ER3 don't directly be regulated by EFD protein,it may have several genes between them.5.We used the promoter of A9 and ATA7 which were specifically expressed in the tapetum of anther,to overexpress gene ER3.The expression level of ER3 gene was detected by real-time PCR,to judge if the plants were valid overexpression transgenic plants,then taking a close observation to the development process in transgenic plants.