黄瓜耐冷生理机制、分子标记及相关基因分离的研究
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摘要
低温是设施栽培在冬春季节经常遇到的逆境胁迫，作为主栽蔬菜，黄瓜(Cucumis sativus L．)的冷敏性已成为其在保护设施内正常生长的主要限制因子，因此如何提高黄瓜耐冷性便成为目前黄瓜保护地栽培专用品种选育的研究重点，而正确理解黄瓜的耐冷机制则是耐冷育种的基础。本文从生理机制、分子标记及相关基因片段分离等方面进行了研究，以期进一步明确黄瓜的耐冷机制，并获得与耐冷性紧密连锁的分子标记，主要作了如下研究：
1 黄瓜耐冷生理机制研究
研究了栽培黄瓜在15℃低温下的发芽能力，并得到了两个具有明显差异的品种“北京截头”和“长春密刺”，以这两个品种为主要试材，比较研究了黄瓜种子发芽期间低温(15℃)对水解酶(包括蛋白酶和脂肪酶)活性的影响以及冷胁迫(7℃)下Ca~(2+)及其螯合剂EGTA对黄瓜叶片超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)等三种抗氧化酶活性的影响。
水解酶活性实验结果表明：低温减缓了黄瓜种子水解酶活性变化速度，使酶活性高峰的出现延后，从而推迟了种子发芽。低温下冷敏品种水解酶活性明显低于后者，说明黄瓜种子低温发芽能力与水解酶的活性高低有关。根据耐冷品种水解酶活性有急剧上升的现象，可以推测不同基因型黄瓜低温发芽能力存在差异的原因是耐冷品种种子中有水解酶被激活。低温处理早期脂肪酶活性受到抑制，而蛋白酶活性无此现象，说明与蛋白酶相比，脂肪酶对低温更敏感。
Ca~(2+)和EGTA处理的结果表明：CaCl_2处理减缓了而EGTA处理则加速了黄瓜叶片所有3种抗氧化酶活性的下降，说明Ca~(2+)对提高黄瓜耐冷性具有重要作用。在这2个处理和对照实验中，耐冷品种抗氧化酶活性的下降幅度均小于冷敏品种，这可能是两种黄瓜材料耐冷性存在差异的原因；但CaCl_2处理能够缩小而EGTA处理能够扩大这种差距，说明耐冷品种和冷敏品种在Ca~(2+)浓度的调节机制上存在差异。
本文还以田间冷害指数为指标，研究了冷害过程中耐冷性不同的黄瓜品种叶片SOD、CAT和POD活性的变化。结果表明：低温胁迫期间的CAT和POD活性与黄瓜叶片的耐冷性表现一致，SOD活性则与其耐冷性表现相反。低温胁迫后3种抗氧化酶活性均降低，叶片表现出明显的冷害症状，但此时耐冷性强的品种仍具有较高的
As a environment stress, low temperature is often suffered in the protected field at winter and spring, and the chilling sensitivity of cucumber, a major vegetable cultivated in protected condition, has become the main factor disturbing its normal growth, so how to improve the chilling tolerance of cucumber has been the research focus on special breeding for protected growth of cucumber. In this dissertation, the physiological mechanisms, molecular markers and related genes isolation of chilling tolerance in cucumber were studied, in order to deeply understand the mechanism of chilling tolerance and get the molecular markers tightly linking to this trait. The follows were researched:
1 Studies on the physiological mechanisms of chilling tolerance in cucumber
The germination ability at low temperature of 15℃ of cucumber cultivars were measured and two cultivars, "Beijing jietou" and "Changchun mici", with significant difference in this trait were identified. Using the two cultivars as main experimental materials, we studied the effects of low temperature (15℃) on the hydrolases activities, including proteases and lipases, during cucumber seeds germination and the effects of exogenous Ca~(2+) and its chelator EGTA on the activities of three antioxidative enzymes, SOD, CAT and POD, in cucumber leaves during chilling stress(7℃).
The results of hydrolases activities experiments revealed that the low temperature slowed down the change rate of hydrolases activities of cucumber seeds and postponed the appearance of their peeks, and then delayed the germination. The activities of chilling sensitive cultivar the former were obviously less than those of chilling tolerance cultivar, which suggested that the ability to germinate under low temperature in cucumber was related to the hydrolases activities. According to the suddenly increment of activity only presenting in chilling tolerance cultivar but not in the sensitive one, we can deduced that the reason for the difference of germination ability under low temperature between different genetic types of cucumber was that there are hydrolases to be activated in chilling tolerance seeds. At the early stage of low temperature treatment, the activity inhibition of was
1 黄瓜耐冷生理机制研究
研究了栽培黄瓜在15℃低温下的发芽能力，并得到了两个具有明显差异的品种“北京截头”和“长春密刺”，以这两个品种为主要试材，比较研究了黄瓜种子发芽期间低温(15℃)对水解酶(包括蛋白酶和脂肪酶)活性的影响以及冷胁迫(7℃)下Ca~(2+)及其螯合剂EGTA对黄瓜叶片超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)等三种抗氧化酶活性的影响。
水解酶活性实验结果表明：低温减缓了黄瓜种子水解酶活性变化速度，使酶活性高峰的出现延后，从而推迟了种子发芽。低温下冷敏品种水解酶活性明显低于后者，说明黄瓜种子低温发芽能力与水解酶的活性高低有关。根据耐冷品种水解酶活性有急剧上升的现象，可以推测不同基因型黄瓜低温发芽能力存在差异的原因是耐冷品种种子中有水解酶被激活。低温处理早期脂肪酶活性受到抑制，而蛋白酶活性无此现象，说明与蛋白酶相比，脂肪酶对低温更敏感。
Ca~(2+)和EGTA处理的结果表明：CaCl_2处理减缓了而EGTA处理则加速了黄瓜叶片所有3种抗氧化酶活性的下降，说明Ca~(2+)对提高黄瓜耐冷性具有重要作用。在这2个处理和对照实验中，耐冷品种抗氧化酶活性的下降幅度均小于冷敏品种，这可能是两种黄瓜材料耐冷性存在差异的原因；但CaCl_2处理能够缩小而EGTA处理能够扩大这种差距，说明耐冷品种和冷敏品种在Ca~(2+)浓度的调节机制上存在差异。
本文还以田间冷害指数为指标，研究了冷害过程中耐冷性不同的黄瓜品种叶片SOD、CAT和POD活性的变化。结果表明：低温胁迫期间的CAT和POD活性与黄瓜叶片的耐冷性表现一致，SOD活性则与其耐冷性表现相反。低温胁迫后3种抗氧化酶活性均降低，叶片表现出明显的冷害症状，但此时耐冷性强的品种仍具有较高的
As a environment stress, low temperature is often suffered in the protected field at winter and spring, and the chilling sensitivity of cucumber, a major vegetable cultivated in protected condition, has become the main factor disturbing its normal growth, so how to improve the chilling tolerance of cucumber has been the research focus on special breeding for protected growth of cucumber. In this dissertation, the physiological mechanisms, molecular markers and related genes isolation of chilling tolerance in cucumber were studied, in order to deeply understand the mechanism of chilling tolerance and get the molecular markers tightly linking to this trait. The follows were researched:
1 Studies on the physiological mechanisms of chilling tolerance in cucumber
The germination ability at low temperature of 15℃ of cucumber cultivars were measured and two cultivars, "Beijing jietou" and "Changchun mici", with significant difference in this trait were identified. Using the two cultivars as main experimental materials, we studied the effects of low temperature (15℃) on the hydrolases activities, including proteases and lipases, during cucumber seeds germination and the effects of exogenous Ca~(2+) and its chelator EGTA on the activities of three antioxidative enzymes, SOD, CAT and POD, in cucumber leaves during chilling stress(7℃).
The results of hydrolases activities experiments revealed that the low temperature slowed down the change rate of hydrolases activities of cucumber seeds and postponed the appearance of their peeks, and then delayed the germination. The activities of chilling sensitive cultivar the former were obviously less than those of chilling tolerance cultivar, which suggested that the ability to germinate under low temperature in cucumber was related to the hydrolases activities. According to the suddenly increment of activity only presenting in chilling tolerance cultivar but not in the sensitive one, we can deduced that the reason for the difference of germination ability under low temperature between different genetic types of cucumber was that there are hydrolases to be activated in chilling tolerance seeds. At the early stage of low temperature treatment, the activity inhibition of was
引文
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