【作者】 郭爱；

【导师】 郑彩霞；

【作者基本信息】 北京林业大学 ， 植物学， 2013， 博士

【摘要】 油松是我国特有的造林和用材树种,也是重要的园林观赏树种。油松胚珠发育分子调控机制的研究对于油松产种及人工制种工作有重要的理论和实践意义。20世纪90年代在辽宁兴城油松种子园发现了油松雌性不育株,前期工作证明雌性不育系不育的原因是胚珠休眠后雌配子体游离核有丝分裂停止。SLOW WALKER2(SWA2)基因是拟南芥突变体swa2的突变基因,该基因缺乏可导致拟南芥雌配子体有丝分裂停止,最终表现为雌性不育。油松不育系雌配子体游离核旺盛分裂期游离核分裂停止的现象与swa2表型非常相似。本研究旨在探讨SWA2基因在油松雌配子体游离核分裂中表达的阶段特异性、组织特异性及其在可育、不育系胚珠相应发育时期的表达差异。从针叶中提取油松的DNA,在油松中扩增出拟南芥SWA2同源基因(PtSWA2)的片段,之后进行测序得到该目的片段的序列。通过比对,该目的片段与SWA2基因cDNA序列具有高度的一致性,不含有内含子,所以利用该基因序列信息和NCBI GenBank中相关序列信息设计特异性引物,进行反转录PCR,获得油松PtSWA2的EST。测序结果分析表明得到的片段为目的基因的同源片段。RT-PCR和实时定量PCR结果显示,在可育系胚珠中,PtSWA2在休眠期后雌配子体游离核分裂初期、旺盛分裂期、分裂后期和细胞化时期均表达,且在游离核旺盛分裂期表达量最高。在不育系胚珠中,该基因在雌配子体游离核分裂的初期、旺盛期和细胞化时期的表达量分别下调了6%、6.9%和11.7%。该基因在可育不育系胚珠中表达的产物也有明显差异。荧光原位杂交结果显示胚珠雌配子体游离核旺盛分裂时期,PtSWA2荧光信号出现在位置为紧贴胚珠的珠鳞组织；雌配子体游离核细胞化时期,PtSWA2信号荧光出现的位置为雌配子体外围紧贴雌配子体的细胞(绒毡层)。本文首次从油松中获得PtSWA2的DNA片段和EST,并对其在胚珠雌配子体发育中的表达进行研究,得到结论如下：(1)PtSWA2在油松胚珠雌配子体发育的不同阶段均表达,且其表达具有阶段特异性和组织特异性。(2)在可育系与不育系油松胚珠雌配子体发育的不同阶段PtsWA2的表达具有明显差异：PtsWA2在可育系胚珠雌配子体发育不同时期的表达量均明显高于其在不育系胚珠雌配子体发育相应时期的表达量。在可育系胚珠雌配子体游离核分裂期出现约2kb大小的扩增产物在不育系雌配子体发育相应时期未检测到；在可育系胚珠细胞化时期未检测到扩增产物,但在同一发育时期的不育系胚珠中却检测到了约2kb大小的扩增产物。(3)根据荧光原位杂交结果推测该基因与胚珠营养组织细胞代谢有关。更多还原

【Abstract】 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.更多还原