苹果果皮光调控相关基因cDNA-AFLP差异表达分析
摘要
光是植物生长发育的重要环境因子之一,光条件的改变影响着植物抗氧化能力、果实着色等。苹果(Malus domestica)是我国重要的栽培果树之一,其套袋已成为改变果实外观品质的一个重要手段,套袋果解袋后表皮色泽发生明显的改变。又有研究表明,光条件的改变可引起植物分子表达水平的差异。
本试验以套袋遮光和自然光条件下“嘎啦”苹果果皮为试材,应用cDNA-AFLP方法分析不同光条件下苹果果皮差异表达的基因,为进一步准确、全面地研究苹果果皮光保护机制及各种生理现象的内在机理提供理论依据。主要研究结果如下:
1.通过cDNA-AFLP技术对套袋和非套袋苹果果皮cDNA进行筛选、分析,初步获得差异表达的基因片段。
2.对差异基因进行扩增、回收、克隆、测序及同源序列比对,同源性结果包括:β-1,3-葡聚糖合成酶,转运RNA,酿酒葡萄基因文库序列,烟草细胞死亡过程中过氧化氢调控基因。
3.通过半定量分析,结果表明非套袋和套袋条件下果皮表达量存在差异,在非套袋果皮上tRNA-Leu基因表达显著高于套袋;与套袋相比,解袋后表达量有所回升。
4.对不同光条件下果皮游离氨基酸含量进行测定,结果表明:自然光下苹果果实阳面果皮中亮氨酸含量明显高于套袋遮光的果皮,解袋后果皮亮氨酸含量有所回升,与半定量表达分析结果一致。
Light is one of the important factors which affect various aspects of fruit, such as antioxidant capacity, fruit coloring. Bagging is an effective measure to shade light, which could improve fruit appearance quality. The fruit coloration was significantly changed after removing shades. The change of light condition might lead to a clear change in the fruit epidermis with regulation of gene expression.
To understand response mechanism of fruit peel to different light t conditionshe on mRNA level and provide some evidence for inner operating mechanism of physiological phenomena and light protection, cDNA-amplified fragment length poly-morphism experiments were used to identify differentially expressed genes from peels of bagged and unbagged apples. The results were as follows:
1. The peels of apple were taken as the material under different light condition. The cDNA-AFLP method was used to analyze the gene expression characteristics between shading and natural light condition in apple peel, and then the different expression genes were separated preliminarily.
2. The primary fragments were used to secondly amplificated, Then transcript-derived fragments were isolated, cloned and sequenced. The resualts of sequence homology analysis include: Nicotiana alata putative 1,3-beta-glucan synthase mRNA, Nicotiana tabacum tRNA-Leu gene, whole genome shotgun sequence of Vitis vinifera, Nicotiana tabacum cDNA-AFLP fragment sequence.
3. After analyzing of the sequences, the fragments of tRNA-leu were uesed to further investigate. Its expression of mRNA was monitored by using a semi-quantitative RT-PCR-based protocol under different light condition. The transcript level of tRNA-leu was significantly decreased in the peel of bagging apples and showed a clear increase after the bag was removed.
4. The free amino acids in the peels were detected. The results showed that shading with bagging reduced the content of free aspartic acid in apple peels, and the content of free aspartic acid was clearly back after the bags removed, which was agreeable with the transcript level of tRNA-leu
本试验以套袋遮光和自然光条件下“嘎啦”苹果果皮为试材,应用cDNA-AFLP方法分析不同光条件下苹果果皮差异表达的基因,为进一步准确、全面地研究苹果果皮光保护机制及各种生理现象的内在机理提供理论依据。主要研究结果如下:
1.通过cDNA-AFLP技术对套袋和非套袋苹果果皮cDNA进行筛选、分析,初步获得差异表达的基因片段。
2.对差异基因进行扩增、回收、克隆、测序及同源序列比对,同源性结果包括:β-1,3-葡聚糖合成酶,转运RNA,酿酒葡萄基因文库序列,烟草细胞死亡过程中过氧化氢调控基因。
3.通过半定量分析,结果表明非套袋和套袋条件下果皮表达量存在差异,在非套袋果皮上tRNA-Leu基因表达显著高于套袋;与套袋相比,解袋后表达量有所回升。
4.对不同光条件下果皮游离氨基酸含量进行测定,结果表明:自然光下苹果果实阳面果皮中亮氨酸含量明显高于套袋遮光的果皮,解袋后果皮亮氨酸含量有所回升,与半定量表达分析结果一致。
Light is one of the important factors which affect various aspects of fruit, such as antioxidant capacity, fruit coloring. Bagging is an effective measure to shade light, which could improve fruit appearance quality. The fruit coloration was significantly changed after removing shades. The change of light condition might lead to a clear change in the fruit epidermis with regulation of gene expression.
To understand response mechanism of fruit peel to different light t conditionshe on mRNA level and provide some evidence for inner operating mechanism of physiological phenomena and light protection, cDNA-amplified fragment length poly-morphism experiments were used to identify differentially expressed genes from peels of bagged and unbagged apples. The results were as follows:
1. The peels of apple were taken as the material under different light condition. The cDNA-AFLP method was used to analyze the gene expression characteristics between shading and natural light condition in apple peel, and then the different expression genes were separated preliminarily.
2. The primary fragments were used to secondly amplificated, Then transcript-derived fragments were isolated, cloned and sequenced. The resualts of sequence homology analysis include: Nicotiana alata putative 1,3-beta-glucan synthase mRNA, Nicotiana tabacum tRNA-Leu gene, whole genome shotgun sequence of Vitis vinifera, Nicotiana tabacum cDNA-AFLP fragment sequence.
3. After analyzing of the sequences, the fragments of tRNA-leu were uesed to further investigate. Its expression of mRNA was monitored by using a semi-quantitative RT-PCR-based protocol under different light condition. The transcript level of tRNA-leu was significantly decreased in the peel of bagging apples and showed a clear increase after the bag was removed.
4. The free amino acids in the peels were detected. The results showed that shading with bagging reduced the content of free aspartic acid in apple peels, and the content of free aspartic acid was clearly back after the bags removed, which was agreeable with the transcript level of tRNA-leu
引文
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