【作者】 刘杰；

【导师】 饶力群； 龙桂友；

【作者基本信息】 湖南农业大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 周期性的柑桔冻害严重制约了世界各国柑桔业的发展，各柑桔生产国一直致力于通过抗寒育种来提高柑桔生产的抗冻害能力，柑桔抗寒机理的研究是抗寒育种的基础性工作，然而这方面的深入研究进展缓慢。本试验以抗寒的枳(Poncirus trifoliata Raf．)和抗寒较弱的宫本／枳砧(Citrus reticulata Blanco cv．Miyamoto)品种2年生盆栽苗为试材，模拟柑桔越冬时气温低、土温高的环境，在生长期进行低温处理，研究低温胁迫下生长期枳和宫本苗伤害的生理生化变化及其致死低温，采用蛋白质组学研究手段探讨柑桔在接近致死和完全致死的低温胁迫下特异蛋白质的表达。试验结果如下： 1、用棉絮包裹根颈以下整个盆栽钵，可有效地保持根际土壤温度，—6℃处理20min至80min比不包裹的对照提高土温6℃-9.5℃。 2、在棉絮包蔸使根系不受冻的情况下，对2年生枳和宫本盆栽苗分别进行-6℃10min～50min及-6℃10min～30min处理时，叶片电解质外渗率小于25％，过氧化氢酶和过氧化物酶活性下降，脯氨酸和丙二醛含量变幅不大，叶片不表现冻害；当处理时间分别为60min和40min左右时，电解质外渗率大于50％，过氧化氢酶和过氧化物酶活性达最大值，丙二醛含量达最大值，枳叶片脯氨酸含量出现最大值，宫本叶片脯氨酸出现最小值，叶片受冻，干枯脱落。 3、枳和宫本耐受低温的临界点分别为—6℃处理60min和—6℃处理40min左右。 4、获得了对照、接近致死(枳-6℃50min，宫本-6℃30min)和完全致死(枳-6℃80min，宫本-6℃60min)低温处理的枳和宫本叶片蛋白质银染双向电泳图谱，图谱清晰，蛋白质斑点均匀分散。 5、用PDQuest 7.2分析软件处理银染双向电泳图谱，在枳叶片蛋白质银染双向电泳图谱中，能分辨的蛋白质斑点数最多的为378个，以对照的蛋白质2—DE图谱为参考胶，-6℃处理50min诱导出7个新蛋白质点，有4个蛋白质点消失，上调表达(表达量相差2倍以上)蛋白质点148个，下调表达(表达量相差2倍以上)蛋白质点5个；-6℃处理80min诱导出13个新蛋白点，有4个蛋白质点消失，上调表达蛋白质点73个，下调表达蛋白质点23个。在宫本叶片蛋白质银染双向电泳图谱中，能分辨的蛋白更多还原

【Abstract】 The world citrus industry has been influenced seriously by the injury of periodically low temperature. Much effort has been made to improve the cold tolerance of citrus crop through various breeding methods, however, up to date little progress has been made since the mechanism of citrus cold tolerance has been remaining unknown. In the present study, some biochemical changes and functional proteins induced by cold stress in growth season were investigated in two-year-old potted plants of trifoliate orange (Poncirus trifoliata Raf.), which was the most cold-tolerant among citrus species, and Miyamoto satsuma mandarin (Citrus reticulata Blanco cv. Miyamoto), less cold-tolerant than the former. The results were as follows:(1) The soil temperature in the pots wrapped with cotton was 6°C to 9.5°C higher than that in bald pots.(2) No injury symptom appeared when the potted plants were treated with -6°C 10 min to 50 min for trifoliate orange and -6°C 10 min to 30 min for Miyamoto satsuma mandarin, respectively, with the electrolyte leakage less than 25%, activities of POD and CAT declining and contents of Pro and MDA stable changing. However, obvious injury symptom was observed when the potted plants were treated with -6°C 60 min for trifoliate orange and -6°C 40 min for Miyamoto satsuma mandarin, respectively, with the electrolyte leakage more than 50%, POD and CAT activities and MDA content reaching their maximums, Pro content reaching maximum in trifoliate orange but minimum in Miyamoto satsuma mandarin.(3) The lethal temperatures of trifoliate orange and Miyamoto satsuma mandarin were -6°C for 60min and -6°C for 40min, respectively.(4) A high quality 2-DE profile was obtained. The proteins were separated very well in both dimensions.(5) Employing PDQuest 7.2 software to analyze 2-DE gels, up to 378 protein spots were更多还原