The Expression Of SLOW WALKER2during The Female Gametophyte Development Of Pinus Tabulaeformis

Pinus tabulaeformis is a special species in China, which can be used as timber trees, ornament trees and forest trees. Research on the molecular regulation mechanism of ovule development of P. tabulaeformis has theoretical and practical significance for producing and acquiring seeds. In1990s, female sterile P. tabulaeformis was discovered in Xing Cheng Seed Orchard, Liao Ning province. It has been convinced on base of previous work that the sterile phenomenon is due to the suspension of the free nucleus division of female gametophyte after dormancy. SLOW WALKER2(SWA2) is the mutation in Arabidopsis thaliana mutant swa2. Lack of this gene, mitosis of female gametophyte stopped and finally led to the female sterility of the mutant. However, the reason why the free nucleus division of female gametophyte arrested has not yet been quite understood. This phenomenon has great similarity with suspension of the free nuclei division of female gametophyte during free nuclei mitosis in P. tabulaeformis. In this research, the expression of SWA2homologue in fertile ovules and the differential expression between the fertile and sterile ovules of P. tabulaeformis are discussed.Genome DNA was extracted from needles of normal P. tabulaeformis. SWA2homologue (PtSWA2) fragment and an EST were isolated and analyzed with BLAST and ORF finder. The sequenced target gene fragment has high similarity with that of SWA2. There is no intron contained in the DNA fragment. Specific primers for RT-PCR designed according to the sequence of the amplified DNA fragment and relevant bioinformation. EST of PtSWA2was isolated.In the fertile ovules, RT-PCR and RT-qPCR shows the PtSWA2expressed in the beginning of the free nucleus division, vigorous division of free nucleus division, the end of the free nucleus division and cellularization. The highest expression exists in the free nucleus vigorous division of female gametophyte. In the sterile ovules, the expression of PtSWA2has been down regulated by6%,6.9%and11.7%respectively. The products of PtSWA2in fertile and sterile ovules may be different according to the RT-PCR result.The signal of fluorescence in situ hybridization (FISH) results show the gene may expresses respectively in the ovule scale during vigorous nucleus division and in tapetum cells during cellularization of female gametophyte.Fragments of a DNA and an EST of PtSWA2were isolated for the first time. The expression of the gene has been studied during female gametophyte development. It is concluded that (1) PtSWA2continuously expresses in stages of female gametophyte development. The expression is tissue-specific and stage-specific.(2) The expression of PtSWA2in fertile ovules is different with that in sterile ovules.(3) According to the result of FISH, PtSWA2may have some relation with the nutrition metabolism during ovule development.