Study On Tissue-Specific Expressional Gene Of Female Gametophyte Development At Free Nuclei Mitosis Stage In Pinus Tabulaeformis Carr.

Gene regulational expression specializes in tissue. To investigate the expression differences of genes in haploid and diploid tissues of Pinus tabulaeformis Carr. ovule during female gametophyte development, female gametophytes and the adjacent diploid tissues were isolated from ovules at free nuclei mitosis stage using laser microdissection (LMD). In vitro linear RNA amplification were employed on trace RNAs, which were extracted from these two tissues, and it was obtained that 4.70μg RNA from female gametophytes and 6.08 μg RNA from adjacent diploid tissues. CDNA library construction from the RNA generated two related libraries with titers of 1.71 × 107 pfu/mL and 1.81 × 107 pfu/mL. A total of 156 ESTs were acquired from 227 randomly sequenced clones in libraries. BlastN search showed 62.82% of the ESTs exhibited significant identity to sequences in dbESTs of coniferous species. BlastX alignment demonstrated that 76.28% of the ESTs were categorized as unknown gene transcripts; only 23.72% could be annotated. Ten unigenes were annotated with function-known proteins, among these unigenes, about seven were refer to specific expressional genes from female gametophyte such as ribosomal protein L7a, ubiquitin-like protein 5, rab-type small GTP-binding protein, etc.; and metallothionein-like protein uniquely came from adjacent diploid tissues, while dehydrin 4 protein and DnaJ-like protein are both expressed in female gametophyte and adjacent diploid tissues.Gene expression of P. tabulaeformis ribosomal protein L7a (PtRPL7a) was analyzed using fluorescence in situ hybridization (FISH). It was found that, in fertile ovule of P. tabulaeformis, PtRPL7a worked in the whole ovule at the early stage of free nuclei mitosis; at the middle stage, PtRPL7a only expressed inside the female gametophyte; till the later stage, the expression of PtRPL7a appeared in diploid tissue adjacent to female gametophyte. On the contray, no PtRPL7a expression was detected in infertile ovule of P. tabulaeformis during free nuclei mitosis period, and we speculated that lack of this gene could lead to ovule infertility of P. tabulaeformis.Conclusively, this study described a new method based on LMD and In vitro linear RNA amplification, which could be employed in analysis of tissue-specific expressional gene of P. tabulaeformis ovule development. Using this method, we successfully captured pure female gametophyte and adjacent diploid tissues with high quality RNA from ovule, and found several genes that work specifically in female gametophyte or adjacent diploid tissue during ovule development of P. tabulaeformis thus providing interesting insights. And it was confirmed that expression of PtRPL7a showed spatio-temporal specificity in ovule development, this gene played an important role in ovule infertility of P. tabulaeformis.