受细胞分裂素调控的叶片衰老相关基因的分离

Cytokinins have been shown to be able to delay leaf senescence, but the mechanism underlying anti-senescence is at present far from clear. In this study, the higher water plant Spirodela polyrrhiza was used as experimental material to investigate the mechanism(s) of cytokinins in delaying senescence. First, it was found that excised half-fronds floating on a medium containing only inorganic salts began to senesce in four days when they were cultivated under long-day conditions, but did not senesce within more than two months when they were cultivated in the darkness. If a cytokinin was included in the medium, the half-fronds cultivated under long-day conditions not only lived for more than two months, but also showed increased physiological activities. Therefore, cytokinins were proved to be necessary for the maintaining of normal physiology of mature fronds (leaves) and a new experimental system for investigating the mechanism of cytokinin involved in senescence was established. Then, through differential display of RT-PCR products a gene was found, whose expression decreased in senescing tissues compared to the green tissues. After cDNA cloning and homology comparison with known genes this senescence-down regulated gene was identified to encode serine:glyoxylate aminotransferase (SGAT), which is involved in the photorespiration pathway. Northern analysis showed that the expression of this gene was induced by light and that its mRNA level was elevated by cytokinin treatment, which had not been reported before. Further experiments were performed and the following results were obtained. After 4 days of cultivation under long-day conditions the extractable SGAT activity in the half-frond tissues was decreased and its substrate serine was accumulated. Exogenous serine added into the medium and with a concentration higher than 5x10-3M induced senescence not only in half-fronds but also in intact plants. MOA, a specific inhibitor of serine biosynthesis, with an appropriate concentration delayed senescence of half-fronds under long-day conditions. These results showed for the first time that the anti-senescence effect of cytokinin may be correlated with photorespiration and suggested that serine is perhaps a signal molecule in the signaling pathway of plant senescence. This would provide a new way of thinking in elucidating the mechanism of cytokinin involved in senescence. In addition, another cDNA was also cloned, which represents one of the rbcS genes in Spirodela polyrrhiza. The analysis of the changes in mRNA level of this gene and its encoding protein level showed preliminarily that cytokinin promoted the expression of this gene on both the transcriptional and the translational level. In the previous work a cDNA encoding a cysteine proteinase was cloned from soybean. Northern analysis of the expression of this gene was also performed and showed the expression was down-regulated during senescence and up-regulated by cytokinin.

延缓叶片衰老是细胞分裂素所具有的重要生理功能之一。因此，研究细胞细胞分裂素的抗衰老机制不仅具有重要的科学意义，而且有益于指导农业生产。本申请项目拟以大豆为材料分离受细胞分裂素调控的、与叶片衰老相关且具有调节功能的基因，期望从基因表达水平上阐明细胞分裂素抗衰老的分子生物学机制。项目的完成将使我们的研究水平处于世界领先地位。