【作者】 袁海英；

【导师】 陈力耕；

【作者基本信息】 浙江大学 ， 果树学， 2005， 博士

【摘要】 草莓(Fragaria ananassa Duch.)是一种经济价值很高的速生草本果树,国内草莓果实的总产量从1998年到2004年六年间翻两番。随着我国加入WTO,果品市场已形成竞争更为激烈的局面。人们对水果品质的要求不断提高,高品质水果的生产已成为我国果业与国际市场接轨的关键。由于草莓果实柔软多汁,缺乏坚硬的保护性外皮;且草莓的成熟期短,成熟过程中品质转化快,加上其外果皮极薄,草莓果实在采后运输、储藏及销售过程中易于遭受生理伤害和病菌侵袭而导致腐烂变质,货架期短;其中细胞壁及细胞膜结构的降解代谢促进了这一过程。细胞膜结构的保持和细胞内区域化结构的完整有助于保持果实的采后品质,磷脂酶D活性的调节在其中起重要作用;然而对于磷脂酶D在草莓果实中的作用却未见相关报道。因此,通过对草莓果实发育成熟过程中磷脂酶D的代谢、调控及分子生物学研究,可为明确磷脂酶D在草莓果实发育成熟过程中的作用机理打下基础,从而有助于设计和提出减少细胞膜伤害、提高草莓采后品质、延长草莓采后储藏期的合适的采收、运输及储藏条件和新方法;与此同时,本研究也有助于了解在非跃变型果实草莓中磷脂酶D在信号传导过程中所起的作用。 本研究以草莓品种Aromas利Seascape为试材,对草莓果实不同发育时期磷脂酶D的活陸变化、磷脂酶D的生化特性、活体条件下磷脂酶D的特性,磷脂酶D的亚细胞学定位及分子生物学特性等进行了初步研究,主要研究结果如下: (1) 磷脂酶D活性测定体系的建立在磷脂酶D生理生化特性的研究中起重要的作用。以草莓果实为试材,优化了磷脂酶D活性测定的底物放射标记法,建立了适合草莓果实组织磷脂酶D制备及活性测定的技术体系:该技术体系的建立为后续进一步探讨磷脂酶D在草莓果实发育成熟和衰老进程中的作用奠定基础。 (2) 对草莓果实不同发育时期磷脂酶D活性进行的测定发现,除白果期外,‘Seascape’品种在其他发育阶段的磷脂酶D活性总体上都要高于‘Aromas’品种。‘Aromas’品种线粒体膜磷脂酶D的活性在果实发育的各个阶段变化不人;微粒体膜磷脂酶D的活性随着果实的发育逐渐升高,并在白果期达到最高。‘Seascape’品种微粒体膜磷脂酶D的活性在幼果期最高,之后则迅速下降至其起始水平的一半左右;而其线粒体膜磷脂酶D的活性在绿果期至绿果膨大期间下降,之后呈增强趋势,并在转色期达到最高。草莓果实发育和成熟阶段膜磷脂酶D活性的变化说明草莓果实的发育成熟与膜磷脂酶D活性之间存在相关性。 (3) 不同发育时期草莓果实磷脂酶D蛋白免疫杂交的结果与活性测定结果呈现出相似的变化趋势;杂交结果显示磷脂酶D蛋白的分子量约为92kDa。 (4) 对草莓果实不同组分磷脂酶D生化特性的研究结果发现,线粒体膜磷脂酶D的适宜pH条件为5.5和6.5,微粒体膜磷脂酶D的适宜pH条件为5和7:酶动力学分析认为,更多还原

【Abstract】 Strawberry (Fragaria ananassa Duch) is one of the highly valuable and important fruit in the world. The whole production of strawberry fruits increases twice during the past six years from 1998 to 2004 in China. After entering into the WTO, the domestic market in China faces the even more severe competition from abroad. Therefore high quality fruits become the key to get into the international market. Strawberry fruits are soft and juicy, but lack of hard shell to protect them. Besides, Strawberry fruit reaches its maturity in short time and then get rotten easily during the process of transportation, storage and marketing, which was accelerated by the catabolic breakdown of cellular structure such as the cell wall and the membrane. Preservation of membrane structure and compartmentalization helps maintain the quality of the fruits, in which the regulation of phospholipase D (PLD) plays an important role. But so far to now, no study of PLD was reported on strawberry. Researches on PLD metabolism, regulation and molecular properties during strawberry fruit development and ripening would be helpful to find new methods and ways to reduce the membrane damage and increase the storage quality and shelf life of strawberry fruits. At the same time, it would also be helpful in finding the potential role of PLD in signal transduction pathway of strawberry fruit, a non-climacteric fruit.The present study examined the roles of PLD in fruit development stages by using strawberry fruits of ’Aromas’ and ’Seascape’. The enzymatic characterization, function in vitro and in vivo, sub-cellular localization, and molecular properties were also included in this study. The main results are as follows:(1) It is very important to establish a proper system for detecting PLD activity in strawberry fruits. After optimizing the radiolabeled methods in PLD activity determination, a proper system was established on the preparation of PLD and the detection of its activity in strawberry fruits. This method will help to elucidate the potential roles of PLD in strawberry fruit ripening and senescence process.(2) PLD activity was detected in different development stages of strawberry fruits. In general, PLD activity was higher in ’Seascape’ at all stages except the mature white stage. There was very little change in mitochondrial PLD activity during development of ’Aromas’ fruits. The microsomal PLD activity increased during fruit development and reached a maximum at the mature white stage, and then declined. PLD activity in the microsomal fraction was the highest at the young immature stage in ’Seascape’, after which it declined to nearly half of its original specific activity. Mitochondrial PLD activity also showed a slight decline at the beginning, however it continued toincrease during further development and reached maximal value at the turning orange stage. These results suggest that PLD activity increases during fruit development and ripening and thus PLD may play a role in strawberry fruit ripening.(3) Immunoblots of PLD protein showed similar results with the activity change during strawberry fruit development stages. The results also showed that no matter mitochondrial or microsomal PLD, all had a molecular mass around 92kDa, which was consistent with most reported plant PLDs.(4) The optimum pH for strawberry mitochondrial PLD were 5.5 and 6.5, while for microsomal PLD 5 and 7. Enzyme Kinetic analysis showed that the activities of both mitochondrial and microsomal PLD follow Michaelis-Menten kinetics. Lineweaver-Burk plots showed that the Km constants were 113.89 and 277.01 for mitochondrial and microsomal PLD, respectively. And the maximum velocity for the microsomal PLD was almost 12 times than that of mitochondrial PLD. PLD activity also got activated at around physiological Ca2+ concentration. Proper amounts of calmodulin improved the activation of Ca2+ towards PLD activity. The results show that strawberry fruit mitochondrial PLD and microsomal PLD have different regulatory properties, which may reflect their different functions.更多还原