【作者】 封碧红；

【导师】 李汉霞；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2001， 硕士

【摘要】 叶片是植物进行光合作用的主要器官，作物产量和品质形成的重要源泉，而衰老是影响叶片功能执行的主要因素。延缓叶片衰老对农业和园艺生产具有重要意义。 叶片衰老是一种PCD过程，受核基因的表达调控，也是植物长期进化过程中形成的适应性反应。叶片衰老过程中伴随着一系列有序事件的发生和复杂的生理生化变化，外施植物激素或内源激素水平极大地影响叶片的衰老进程。深入研究叶片衰老的机理，人为调控叶片衰老具有重要的理论意义和实践价值。随着现代分子生物学理论和技术的迅猛发展，激素合成相关基因、叶片衰老相关基因及其调控序列相继分离克隆，利用基因工程技术抑制内源乙烯产生、促进细胞分裂素（CTK）合成以延缓叶片衰老已在多种作物上获得成功。这些材料的获得为深入研究叶片衰老的分子机理、激素及其基因调控叶片衰老的机制，从而更好地调控叶片衰老提供了有力武器。 本实验首先对获得的转P_SAG12-ipt基因番茄后代进行筛选与纯合，然后利用获得的纯合系统和转anti-EFE番茄纯合系统（B₂），研究不同转化植株的抗叶片衰老特性、两种外源基因调控叶片衰老过程中生理生化变化及其异同，为深入探讨激素延缓叶片衰老的作用机制提供科学依据，并得出以下结果： 1．利用苗期卡那霉素（Km）喷施法和PCR扩增结合，经筛选和分离纯合得到两个具有抗叶片衰老的转ipt株系，表示为ipt1，ipt2。 2．叶绿素、光合效率、内源激素的测定结果表明，P_SAG12-ipt基因具有较明显的叶片衰老表达特异性，转基因植株抗叶片衰老作用显著。其下部叶片的叶绿素含量、光合效率明显地高于对照，而中部与上部未衰老叶片其含量则与对照相当或稍高。ipt基因的导入不仅促进了iPA含量的提高，同时也促进了IAA含量的提高，同一叶位不同时期叶绿素含量、内源激素水平的下降速度比对照缓慢。 3．与转ipt株系相比，B₂叶片的叶绿素含量、光合效率以及变化趋势与华中农业丈学砰士学夕谐戈 其相当，但内源激素iPA的含量比转iPt株系低，而IAA的含量则比 转iPt株系高，同样也具有抗叶片衰老的作用。4.从离体叶片和果实乙烯的释放看，B:乙烯释放量明显低于对照，转iPt 的乙烯释放趋势与对照比较一致，但释放量略低于对照，却远高于BZ 株系。5.嫁接结果表明，转iPt株系的内源激素基本不能向上传导，而B:则有 一定的传导。6.田间性状观察，转iPt与BZ株系的株高、主茎叶数与对照无差异，而 结果数、单株产量高于对照，同一叶位叶片与对照相比推迟衰老一周 左右。更多还原

【Abstract】 Leaf plays important roles in yield and quality of crops for its important function in photosynthesis. Leaf senescence seriously blocks its function. So leaf senescence delaying is of great value in agricultural and horticultural production.As a kind of programmed cell death (PCD), leaf senescence, which was evolved to fit for environment stress, is under the control of nucleus genome. Series physiological and biochemical reactions are accompanied with the course of leaf senescence, and these reactions are seriously affected by endogenous and exogenous hormone level in plant. Further study on the mechanism of leaf senescence will have tremendous value in theory and practice. With the development of molecular biology and biotechnology, several genes also their promoters were identified which are associated with plant hormone synthesis and leaf senescence. Transformations with genes that inhibit ethylene or promote cytokinin production were succeeded in several crops, thus provided materials to research the molecular mechanism of leaf senescence through plant hormones.In this experiment, we firstly got the homozygous lines of ever obtained transgenic tomato plants, which contain the chimeric PSAG12-ipt gene. We compared these homozygous lines with the transformed tomato homozygous lines (B2) containing the anti-EFE gene on characteristics of leaf senescence delaying, and the physiological and biochemical changes caused by the twoforeign genes. The results are as follows:1. Two homozygous lines containing ipt gene designated iptl and ipt2 were obtained by kanamycin spraying test in the seedling stage and PCR amplification.2. The assays of chlorophyll, endogenous hormones and photosynthesis ability suggested that the ipt homozygous lines have the apparent characteristics of delaying leaf senescence and developmentally specific expression. When compared to the control, lower leaves of the homozygous ipt plants had higher chlorophyll content and photosynthesis rate but the same or slightly higher in adult and young leaves. Both the levels of iPA and IAA were promoted due to ipt gene expression. Chlorophyll content and endogenous hormone levels droped more slowly in ipt homozygous line leaves compared to the control in the same position.3. Like the ipt lines, B2 lines (homozygous anti-EFE transformed tomato plants) also had the characteristics of senescence delaying, the same trend on chlorophyll content and photosynthesis rate. As to hormones, 82 lines had higher level of IAA but lower level of iPA than ipt lines.4. Ethylene released from fruits and detached leaves were detected, ipt lines had the same trend with the control but the level was slightly lower, while 82 lines had far less ethylene release than the control.5. Grafting experiments suggested that the endogenous hormones iPA and IAA of ipt lines could not conduct upward but 82 lines could conduct on some degree.6. Investigation in the field showed that transgenic plants of ipt lines or 82 lines had more fruits and heavier fruit. The onset of leaf senescence can be retarded about a week but no difference in height and leaf number when compared to the control.更多还原