Screening And Analysis Of Potential Imprinted Genes In Early Embryogenesis Of Tobacco

There is a maternal control in the early embryogenesis of mammals. But little is known about the form and extent of the parental-origin transcripts involved in the early embryo development in the higher plants. In the plant remote crosses, even no research has been focused on the roles of paternal transcripts played in the early embryogenesis. So we construct a cross system (Hamayan(?)×SR1(?)) to investigate the activity of the paternal genome in the early development stages by detecting cDNA from parents in the hybrid embryos. Based on our observation, some transcripts derived from paternal or maternal parent had already existed even in the zygote stage. These transcripts might be expressed by imprinted or potential imprinted genes. However, nearly no knowledge of biological function of imprinted genes is known in embryognensis of plants. Benefited from the screening in the interspecies hybrid, a potential paternal imprinted gene EB30was found. By Genome walking and hiTAIR, the promoter sequence was obtained. Then we detected the expression pattern, sub-celluar location and imprinting expression of EB30. Also, RNAi transgenic plants were constructed to investigate the biological function of EB30in the plant development. Recently, a new type of cysteine protease inhibitor NtCYS which played an important role in the PCD of the suspensor was found in our lab. To find out the possible function of maternal transcripts in the PCD of the suspensor, we investigated the allelic expression pattern of NtCYS by both an intraspecific cross and an interspecies cross. Our results suggested that NtCYS was likely a maternal imprinted gene, and we confirmed that NtCYS was a maternal-origin gene in in the hybrid of an interspecies cross. Other results also indicated the special effect of the maternal transcripts in the development of the hybrid suspensor. The main results of our work are as follows:1. Establishment of an interspecies cross system:two tobacco species, Nicotiana tabacum var. SRI and N. rustica var. Hamayan were selected for their successful interspecies hybridization. The successful results indicated that the two kinds of genomes were at least compatible between the two species. However, due to the low availability of hybrid embryos (SR1(?)×Hamayan(?)), the hybrid embryos (Hamayan(?)×SR1(?)) were mainly used in later experiments.2. A well-established method in our lab allowed us to isolate enough living sperm cells, zygotes and8-celled embryos for the construction of cDNA pools. Living sperm cells were obtained from SRI and living zygotes,8-celled embryos were obtained from the SRI, Hamayan and their hybrid (Hamayan(?)×SR1(?)) respectively. mRNA were collected by the mRNA DIRECT Micro Kit, and then reversely transcripted and amplified by the Super SMARTTMcDNA PCR Synthesis Kit.3. The detection of allelic expression pattern of genes exrpessed in the interspecies hybrid embryos:ESTs of tobacco were searched through NCBI. Softwares were used to select ESTs with SSR sites and to design primers. Finally,28genes were found to express in all zygote and8-celled embryo cDNA pools of SRI, Hamayan and the hybrid (Hamayan(?)×SR1(?)).The PCR products were separated by PAGE and results showed that eight genes had clearly parental origins. It demonstrated that parental transcripts were differently involved in the zygote development, and the allelic expression pattern of some genes were dynamics during the embryo development. Also, the eight genes were detected in the cDNA pool of sperms, it revealed that the paternal transcripts in zygotes might be inherited from the sperm cells or were the result of de novo transcription in the zygote.4. Clone and analysis of a potentially paternal imprinted gene EB30:EB30were obtained by previous work, it was found to be a paternal expression gene in the zygote of an interspecies hybrid. Bioinformatic analysis showed that EB30contained211amino acids and was obviously hydrophilic. There were homologous sequences of EB30in crops as Ricinus communis, Sorghum bicolor, Oryza sativa, Zea mays and Vitis vinifera. All homologous sequences had some conserved origins, but little knowledge of them was available. Real time PCR showed that EB30expressed widely. Subcellular localization of EB30revealed that it scattered in cytoplasm as tiny granules. It suggested that EB30might locate on some organelles. Through reciprocal cross of wild type plants and transgenic plants carrying GFP fusion protein, EB30was not proved to be a paternal imprinted gene. Moreover, no obvious phenotype was observed during the development of RNAi plants.5. The detection of expression of NtCYS in the hybrid of an intraspecific cross: NtCYS can regulate the PCD of the suspensor in Nicotiana tabacum var. SR1. But no allele with SNP polymorphism was found in11varieties of Nicotiana tabacum. Since there was no allele of NtCYS detected in KY14, reciprocal cross of SR1and KY14was made to construct2-celled embryo cDNA pools of the hybrid. By detecting the relative expression of NtCYS in cDNA pools of the two hybrids, it indicated that NtCYS might be a maternal imprinted gene.6. The detection of relative expression of NtCYS alleles in the hybrid of an interspecies cross:two potential alleles of NtCYS were found in Nicotiana. rustica var. Hamayan.We detected the relative expression between NtCYS from SRI and alleles from Hamayan in the zygote and8-celled embryo cDNA pools of the hybrid (Hamayan(?)×SR1(?)). The result confirmed that maternal transcripts of NtCYS dominantly expressed in the interspecies hybrid. Futhermore, by detecting the TUNEL signal in32-celled embryo of two parents and the hybrid, we found that no only homologous sequences of NtCYS existed in two patrents and the hybrid, but also there was a similar PCD process of the suspensor in all of them.7. Comparision of of suspensor developement among the hybrid of an interspecies cross and two parents:there were obvious differences on the cell numbers of the suspensor between two parents. By comparing the cell numbers of the suspensor among the hybrid and two parents at the32-celled embryo stage, we found that the cell proliferation of the hybrid suspensor was similar with the maternal parent.In conclusion, the paternal genome has been activated and the parental genomes are differently involved in the early embryogenesis as early as at the zygote stage in a tobacco interspecies hybrid. And the paternal transcripts may come from both sperm delivery during fertilization and de novo transcription in zygote. Furthermore, the allelic expression patterns of some parental-origin genes are dynamics during the developmental process. In addition, our work suggestes that NtCYS involed in the PCD of the suspensor may be a maternal imprinted gene. And we confirm that maternal transcripts of NtCYS dominantly express in the interspecies hybrid. It is also confirmed that the cell proliferation in the suspensor of the interspecies hybrid is similar to the maternal parent in some extent. These results indicate the maternal effect on the development of the suspensor of the interspecies hybrid. Interspecies crosses are widely used in the agriculture, and crops can obtain heterosis at early development stage. To know more about the contribution of parental genomes in the embryogenesis of the hybrid will help us to understand molecular mechanism of the heterosis and establishment of bybrid fertility.