论文部分内容阅读
利用Percoll密度梯度离心和多次滤膜过滤分离到适于分子生物学研究的水稻 (OryzasativaL .cv .Guichao2 )生活精细胞。借助RT_PCR和SMART技术构建了水稻精细胞的cDNA文库。初级文库的大小为 1.5 8× 10 6 pfu ,插入片段平均大小为 980bp。 10 3个随机挑选的克隆与DIG标记的水稻幼苗根、叶及二细胞时期花粉、成熟花粉、精细胞和授粉 5～ 7d的子房cDNA探针杂交表明 ,除少数克隆外 ,它们在上述器官中的表达情况很相似。 10个随机挑选的克隆用来测序及分析 ,有 4个克隆含有完整的开放阅读框。 1个克隆与水稻Polyubiquitin (Rubq1)mRNA高度同源。Northern杂交表明它在二细胞时期花粉、成熟花粉中的表达显著少于在根、叶和授粉子房中的表达。另一个克隆的开放阅读框编码与拟南芥 (Arabidopsisthaliana (L .)Heynh .)AtRAD17同源的蛋白。这是第一次正式报道高等植物精细胞cDNA文库的构建 ,也是第一次从高等植物精细胞中分离到其表达的基因。
Rice (Oryzasativa L.cv. Guichao2) sperm cells suitable for molecular biology studies were isolated using Percoll density gradient centrifugation and multiple membrane filtration. A cDNA library of rice sperm cells was constructed by RT_PCR and SMART techniques. The size of the primary library was 1.5 8 × 10 6 pfu and the average insert size was 980 bp. 10 randomly selected clones hybridized with DIG-labeled rice seedlings root, leaf and two-cell stage pollen, mature pollen, sperm cells and ovary cDNA probes from 5 to 7 days of pollination showed that except for a few clones, In the expression of the situation is very similar. Ten randomly selected clones were used for sequencing and analysis, and four clones contained the complete open reading frame. One clone was highly homologous to rice Polyubiquitin (Rubq1) mRNA. Northern blotting showed that it expressed significantly less in two-cell stage pollens and mature pollen than in roots, leaves and pollinated ovaries. The other clone’s open reading frame encodes a protein homologous to AtRAD17 in Arabidopsisthaliana (L.) Heynh.). This is the first official report of the construction of a higher plant sperm cell cDNA library and is the first one to isolate its expressed gene from higher plant sperm cells.