Expression And Regulation Of Non-imprinted Genes Related To Endosperm In Rice

Rice is one of the world-wide food crops, which production accounted for about 30% of the world’s food crops, and more than 40% in China. The endosperm of rice weight more than 90% of unpolished rice.The development status of endosperm directly determine the yield and quality of rice. Assaf Zemac found that some genes only expressed in the endosperm, these genes are organ-specific. The endosperm-specific genes can be divided into imprinted genes and non-imprinted genes. The role and regulation of imprinted genes are related to seed size. The role and regulation of non-imprinted endosperm-specific genes need to further study.Assaf Zemac made the rice endosperm sequencing analysis and found 165 genes which expressed specifically in endosperm. Based on the genes and the difference of SNP of indica and japonica in Pubmed website, primers are designed to amplify the 22 non-imprinted genes. In this study, indica 9311 and japonica Nipponbare are crossed reciprocally, endosperm RNA of hybrid seeds 3 and 5 days after pollination are for expression study. Sequencing methods are used to determine the endosperm-specific non-imprinted genes. And RNAs from blades are further validated these non-imprinted genes are expressed endosperm-specifically.As known that endosperm-specific expression of imprinted gene is regulated by DNA methylation.However regulation of non-imprinted genes is still unknown.The regulation of endosperm-specific non-imprinted genes are speculated that they may share the same way with the imprinted genes.which means that DNA methylation may also involved in. The indica-japonica hybrid seeds in early germination are treated with demethylation regent 5-azaC. As known that these endosperm-specific non-imprinted genes in the embryo are silent, after removal of DNA methylation, will the gene could be re-expressed in the embryo? The germinating leaves after treatment are taken for research material, non-imprinted gene expression is analyzed whether the gene expression changed. Specific results are as follows:1. Endosperm RNA of 5 days is taken as a template, four endosperm-specific expression genes are screened, and its endosperm specificity is proved. Aiming at 165 genes provided from documents, according to the annotation of the gens, we select the Transcription factor as the main research direction, initially 18 genes are screened.According to SNP differences between indica and japonica and limits of primers, and we identify four non-imprinted genes as of the target gene in this experiment. The result show that these four genes are only expressed in the endosperm, and silent in the blade.2. Endosperm RNA of 3 days and 5 days are taken as template for PCR, the results show that LOC_Os05g34310.1, LOC_Os07g0842 and LOC_Os02g12310.1 exhibit no temporal specificity, the expression don’t change with time. LOC_Os01g24460.1 shows temporal specificity. The expression of most genes has nothing to do with the developmental stages, only the expression of LOC_Os01g24460.1 gene is affected by developmental stages. LOC_Os01g24460.1 gene in the developing endosperm of 3 days show complete inhibition of thymidine in bimodal curve of Sequence diagram, while in 5 days endosperm, it expressed normally.3. After the demethylating agent 5-azaC treating with mature hybrid seeds between Nipponbare and 9311, we find that rice phenotype changed. PCR experiments show that only LOC_Os07g08420 and LOC_Os02g12310.1 expressed in the leaves after 5-azaC treatment, and exhibiting thymine inhibited in bimodal curve of Sequence diagram. It preliminary confirms that the DNA methylation involved in the regulation of non-imprinted genes in rice endosperm. DNA methylation regulate only part of the non-specific imprinted genes. The study find that the germination rate generally low in the scenario of 5-azac treating seeds for 3 days-9 days. In this study we select the processing blade RNA of NIP x 9311,9311 x NIP treated for 3 days and 5 days, detecte the changes in the expression of target 4 genes,and find LOC_Os07g08420 and LOC_Os02g1310.1 gene expression activated, LOC_Os05g34310.1 and LOC_Os01g24460.1 gene remain silent,which means the silence of LOC_Os05g34310.1 and LOC_Os01g24460.1 in embryo is irrelevant in the regulation DNA methylation.4. Treating another japonica rice Balilla with 5-azaC shows only LOC_Os02g12310.1 exhibits thymine suppression in bimodal curve of Sequence diagram, LOC_Os07g08420 shows no significant change. After Barilla xNipponbare and 9311×Nipponbare mature seeds treating with 5-azaC, extracting RNA from the germination leaves, LOC_Os07g08420 gene shows no inhibition of bimodal thymine in Barilla xNipponbare, and exhibits inhibition against thymine in Nipponbare×9311. LOC_Os07g08420 gene probably exhibits differently because of different materials, indicating that genetic diversity of the same gene in different materials. Activatity and the expression of some genes have a material-specificity.In conclusion, this study shows the pattern of non-imprinted gene expression in endosperm share the same diversity with the imprinted genes.Not only the epigenetic regulation by DNA methylation is involved, but also other unknown ways participated. And the expression of these genes is also affected by the material and developmental stages, and therefore is a more complex regulation. This study selects only four genes.InP order to get more scientific results, we need to expand the number of genes for selection. Aiming at the way of regulation by DNA methylation, and further analysis of the characteristics of its regulation is needed.