Analysis Of Gamete And Early Proembryo Transcriptome In Tobacco

The female gamete and embryo in angiosperm is embedded deeply in ovule tissues. The inaccessibility of egg cell and early embryo greatly limits the investigation for molecular mechanism of fertilization and early embryogenesis. To investigate the transcript profiles of gamete and the reconstruction of transcriptome after fertilization, four cDNA libraries were constructed using isolated sperm, egg, zygote and two-celled proembryo from tobacco respectively. The main results are as follows:1. Isolated tobacco sperm cells, eggs, zygotes and two-celled proembryos were used to constructed cell-type specific cDNA libraries by SMARTTM cDNA library construction kit. Two transcription inhibitors:Actinomycin D and Cordycepin were used to inhibit the modulation of gene expression in response to possible stresses during physical isolation and enzymatic treatment. The titer of each libraries was 1.43×106/ml for sperm cell library,4.42x106/ml for egg cell library,4.4×106/ml for zygote library and 5.4×106/ml for two-celled proembryo library. The average length of insertion fragment in each library was 600bp,600bp,800bp and 700bp, respectively. The percentages of recombination of each library were about 99%. It is the first attempt to construction the egg cell, zygote and two-celled proembryo specific cDNA libraries in dicotyledonous plant. It provides us useful resources to study the mechanism of early embryogenesis and zygotic gene activation in plants. 2. Clones from four un-amplified libraries were randomly picked and were sequenced from their 5’ends in a single run and totally got 8334 EST. Bioinformatics analysis indicates that primary metabolism and protein modification seems low in sperm cell. The proportion of novel transcripts in sperm is higher than other libraries, which were nearly 13% in average. Transcriptional activity and protein metabolism seems higher in the unfertilized egg cell. Especially, ECA1 was found abundant in egg cells. From egg to zygote, their transcription showed distinct differences with 405 EST present 139 genes in common, but zygote and two-celled proembyro share more much common transcripts, with 829 ESTs represent 208 genes. This is also indicated by that the amount of comment transcripts between egg and zygote is similar to that between egg and two-celled embryos. The diversity of transcriptome from egg to zygote implies that the transcriptome was indeed reconstructed in zygote, which may owe to the degrading of transcripts deposited from egg or/and de novo transcripts from zygotic genome. The similarity of transcriptome between zygote and two-celled proembryo suggests that their development is regulated by similar mechanism without intensive transcriptome reconstruction. Histones gradually increased after fertilization and first zygote division suggests that genome of zygote already transcriptional activated. This indicates that zygotic gene activation (ZGA) may occurs in zygote in tobacco, which is early than that in animals.3. In order to investigate the real expression pattern in tobacco, we chose several clusters from each of the libraries and carried out the RT-PCR. Seven novel transcripts were identified as sperm cell-specific since they were found only expressed in sperm cell or also in anther. Such transcripts with no identified biological functions indicate the low representation of male gamete specific gene in database. The transcripts from egg cell were no longer found in zygotes, indicating that partial transcripts deposited in egg were degraded in zygote after fertilization. There were also novel transcripts appears in egg cell only after fertilization. Both the degradation of transcripts deposited in egg after fertilization and the de novo transcripts appearance in zygote, which were the two part of the MZT, we conclude that the maternal to zygotic transition occurs in zygote stage. 4. One of the transcript found de novo expressed after fertilization in zygote was identified as dynamin and called NtDynamin. We got the upstream promoter sequence of NtDynamin by genome walking. After cloned the PNtDynamin into pCambia 1302 vector with different report gene:GUS or eGFP, we transformed it both into arabidopsis and tobacco. We have now gotten the Arabidopsis plant transformed by PNtDynamin::GUS. The expression patterns of PNtDynamin::GUS and PNtDynamin::eGFP both in Arabidopsis and tobacco were investigated and the function analysis of NtDynamin in early embryogenesis is being carried on.