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苹果(Malus×domestica L.)和梨(Pyrus communis L.)果实在低温储存过程中虎皮病的发生与果皮中受乙烯诱导的α–法尼烯合酶基因(AFS)的表达及α–法尼烯的积累密切相关。采用TAIL-PCR技术分别从‘富士’苹果和‘丰产’梨中克隆到了约2 kb的α–法尼烯合酶基因(AFS)启动子序列,并提交至Gen Bank中(登录号分别为KM676083和KM676082)。序列比对发现两启动子同源性仅为48.31%,远低于其下游编码区97%的相似度。顺式作用元件在线预测分析发现二者同时存在乙烯、脱落酸、茉莉酸、水杨酸等激素响应元件,低温、厌氧、病原菌等胁迫响应元件,以及3个节律性表达元件和2个诱导子响应元件。对‘丰产’梨中AFS基因表达模式的分析也证实了部分预测的诱导因素对其表达有调控作用。将‘丰产’梨AFS基因启动子序列进行系列删除,与GUS报告基因构建融合表达载体转化烟草,利用GUS染色方法分析了不同长度启动子的转录活性差异,发现缩短至153 bp的启动子片段仍具有较强的转录活性,且长度在276 bp至573 bp的启动子片段其驱动下游基因转录的活性较其他片段更强。通过后续对各作用元件尤其是乙烯响应元件的研究将有助于进一步从分子水平认识虎皮病的发生机制。
The occurrence of tiger skin disease in apple (Malus × domestica L.) and pear (Pyrus communis L.) fruits during cold storage was related to the ethylene induced α-farnesene synthase gene (AFS) Farnese is closely related to the accumulation. The 2-kb α-farnesene synthase gene (AFS) promoter sequence was cloned by TAIL-PCR from ’Fuji’ apple and ’high yield’ pear respectively and submitted to Gen Bank (accession numbers KM676083 And KM676082). Sequence alignment revealed that the homology of the two promoters was only 48.31%, much lower than the 97% similarity of the downstream coding region. On-line predictive analysis of cis-acting elements revealed that both the hormone-responsive elements such as ethylene, abscisic acid, jasmonic acid and salicylic acid, stress response elements such as hypothermia, anaerobic and pathogenic bacteria, as well as three rhythmic expression elements and two induced Sub-response element. Analysis of the AFS gene expression pattern in the ’high yield’ pear also confirmed that some of the predicted induction factors have a regulatory effect on its expression. The promoter sequence of AFS gene was deleted from the series of high yielding pear and fused with GUS reporter gene expression vector to transform tobacco. GUS staining method was used to analyze the differences in transcriptional activity of different length promoters. The results showed that the promoter fragment shortened to 153 bp Has a strong transcriptional activity, and the length of the 276 bp to 573 bp promoter fragment driven downstream gene transcription activity more than other fragments. The follow-up of the various elements, especially the ethylene response element research will help to further understand the molecular pathogenesis of tiger’s disease.