【作者】 张玉华；

【导师】 薛林宝； 缪旻珉；

【作者基本信息】 扬州大学 ， 蔬菜学， 2004， 硕士

【摘要】 为探讨高温对黄瓜花粉发育及受精过程的影响，本文采用比较方法研究了高温与常温下黄瓜耐热品种JY-4和热敏品种新泰密刺处于单核至双核花粉粒时期的花药及新泰密刺授粉后柱头和子房中Ca²⁺分布、ABA含量及蛋白质合成的情况；同时研究了外源Ca²⁺及ABA处理对高温下黄瓜花药及授粉受精过程的影响，结果如下： Ⅰ经45℃处理1h后两品系花药胞内Ca²⁺水平均有所升高，其中新泰密刺上升较JY-4快，但在温度恢复至28℃后其钙稳态的恢复较慢；45℃处理1h后两品系花药内源ABA含量均显著提高，新泰密刺上升幅度比JY-4大，且恢复至28℃后下降速度也较快；应用蛋白质双向电泳技术分析了热激前后可溶性蛋白质合成的变化，高温处理未能造成此期黄瓜花药新蛋白质的合成，但蛋白MW70KD（PI5.7）、MW45.5KD（PI5.6）的表达经热激后明显增加，且在JY-4中的表达水平显著高于新泰密刺。上述结果表明，Ca²⁺和ABA均参与了黄瓜花药对高温胁迫的反应调节；黄瓜单核至双核花粉粒时期花药中热激蛋白的合成至少已被部分抑制，这可能是该阶段黄瓜花药对高温敏感的重要原因。 Ⅱ28℃下，黄瓜（新泰密刺）授粉后柱头中的Ca²⁺主要分布在细胞间隙中，经45℃处理后细胞间隙和胞内Ca²⁺水平均显著升高，胞内外Ca²⁺浓度梯度逐步丧失；45℃处理1h后新泰密刺柱头和子房中内源ABA含量显著提高；高温处理后新泰密刺柱头和子房中产生了一些新的蛋白质，如柱头中的MW26.0KD（PI5.4）、MW90.0KD（PI5.1）、MW54.0KD（PI4.6）、MW41.0KD（PI6.2），子房中的MW90.0KD（PI5.2）、MW61.0KD（PI5.4）、MW48.0KD（PI6.4）、MW40.5KD（PI4.9）：另有一些蛋白质与常温相比表达量明显增加，如柱头中的MW67.0KD（PI5.8）、MW56.5KD（PI5.8），子房中的MW70.0KD（PI5.7）、MW57.0KD（PI5.7）。 上述结果表明，Ca²⁺和ABA信号系统均参与了授粉后黄瓜雌性器官对高温胁迫扬州人学硕熟学位论文 的反应调节，最终可能导致基因表达发生变化。m用!.smmolL‘的cacl:和1.m mc)IL‘的ABA喷施JY一4和新泰密刺单核至双核 花粉粒时期的花药后置于45Oc和28“c下处理。外源caZ+能降低高温下黄瓜花药 的饱内ca2+水平并提高花粉萌发率，而ABA的作用与此相反。外源ca2+和ABA 处理均能显著提高高温下黄瓜花药内源ABA的水平，并产生新的蛋白质，如喷 施外源caZ十后新泰密刺和JY一4花药中ij]新增了蛋白Mw56.oKD（P巧.1），新泰 密刺花药中增加了MW92 .SKD（P15.2几JY一4花药中增加了MW58.OKD（P巧.3）Mw92.5（PIS.5）;外源ABA处理后新泰密刺花药中新增MW46.O‘PI6.。），4花药中新增Mw56.oKD（PIS.l:）以上结果表明，高温下外源ca2+可影响外源ABA和 Y黄瓜花药内ABA信号系统，继而可能改变基因表达，井获得耐热性;亦能影响黄瓜花药内caZ+信号系统脚用1 .smmolL-l的cacl:和1.01 molL’的ABA喷施黄瓜雌花和雄花，正常授粉 后置于45c和28℃下处理，观测柱头和子房I一扫caZ+分布、ABA含量及蛋白质合 成变化情况外源ca2+减少一了高温下黄瓜柱头和花扫另}导组织的胞外ca2+内流， ，的外源ABA的作用不明显。外源ca价和ABA处理均显著降低高温下黄瓜柱头 和子房内源ABA的水平，同时产生新蛋白质。45‘℃下，喷施外源Ca2+和ABA 后子房内均新增蛋白Mw90.OKD‘PIS.1);喷施外源ca2+后子房内新增 MW56.OKD、PIS.0)、MW34.SKD（PI6.O），柱头I匆新增MW35.OKD（PI6.l）、 Mw56.（）KD（PIS.】）;喷施外源ABA后子房内增加MW65.OKD（PIS.4少、 Mw58OKD（P巧.4），柱头内新增Mw63.SKI）（P巧.7）。以_以占果表明，高温下 外源caZ十和ABA均可影响黄瓜雌花内ca2+和ABA信号系统，继而可能导致纂 因表达的变化‘更多还原

【Abstract】 In order to elucidate the effect of heat stress on pollen development and fertilization in cucumber(Cucumis sativus L.), Ca2+ distribution(using potassium antimonite precipitate method), ABA contents and protein synthesis patterns in uninucleate-binucleate-pollen-phase anthers of cucumber heat-tolerant line JY-4 and heat-sensitive line Xintaimici, and in stigma and ovaries of Xintaimici after pollination, were investigated under 45 Cand 28 C. We also studied the effect of exogenous Ca2+and ABA on anthers and stigma , ovaries after pollination of cucumber. The results showed that:I Under 45 C treatment, Ca2+ precipitates increase in pollen and anther cells of both lines, more quickly in Xintaimici than in JY-4. While the temperature recover to 28 C after 45 C stress, the Ca2"1" precipitates quantity drop to the normal level more tardily in Xitaimici anther cells. Endogenous ABA contents in two line anthers also ascend significantly after Ih 45 C treatment, especially in Xintaimici. When the environment return to 28 C, ABA in Xitaimici anthers step down to the control level, while JY-4 anthers can maintain a higher ABA content within 2h. No any additional protein points can be seen in the IEF-SDS-PAGE maps of cucumber anthers in this stage under 45 C stress comparing with 28 C, although the expression of some proteins, such as MW70KD(PI5.7) and MW45.5KD(PI5.6) were upregulated remarkably after heat treatment(even higher in JY-4 than in Xintaimici). The results indicated that both Ca2+ and ABA signals were involved in the response of cucumber anthers to heat-stress. The synthesis of HSPs in cucumber uninucleate-binucleate-pollen-phase anthers were at least in part inhibited under heat treatment, it maybe an important reason why cucumber anther is sensitive to hightemperature during this stage.II Under 45 C treatment, Ca2+precipitates increase in both apoplast and cytoplasm of stigma tissues, and little Ca2+ quantity difference can be seen between apoplast and cytoplasm comparing to the 28 C, under which there are more Ca2+ precipitates in apoplast than in cytoplasm. Endogenous ABA contents in stigma and ovaries also ascend significantly after Ih 45 C treatment. Some additional protein points can be seen in the IEF-SDS-PAGE maps of stigma and ovaries after 45 C stress comparing with 28 C, such as proteins MW26.0KD(PI5.4), MW90.0KD (PI5.1 X MW54.0KD (PI4.6), MW41.0KD (PI6.2) in stigma andMW90.0KD (PI5.2X MW61.0KD (PI5.4), MW48.0KD (PI6.4X MW40.5KD (PI4.9) in ovaries. Furthermore, the expression of proteins MW67.0KD (PI5.8 ) ,MW56.5KD (PI5.8) in stigma andMW70.0KD(PI5.7),MW57.0KD(PI5.7)in ovaries were upregulated remarkably after heat treatment. The results indicated that both Ca2+ and ABA signals were involved in the response of cucumber female reproductive organs to heat-stress and then change the gene expression.IIIUnder 45 C, Exogenous Ca2+ reduce the intercellular Ca2+ of cucumber anthers and increase the pollen germination percentage at high temperature, while the exogenous ABA have a reverse effect. Endogenous ABA contents in two line anthers ascend significantly linked with new proteins formation after exogenous Ca2+ and ABA treatment under 45 C, such as proteinMW56.0KD(PI5.1) in both Xitaimici and JY-4, MW92.5KD (PI5.2) in Xitaimici, MW58.0KD (PI5.3) andMW92.5 (PI5.5) in JY-4 after Ca2+treatment. AndMW46.0 (PI6.0) in Xitaimici, MW56.0KD (PIS. 1) in JY-4 after ABA treatment. The results indicated that Ca2+ and ABA signals can interact each other in cucumber uninucleate-binucleate-pollen-phase anthers under high temperature.IV Ca2+ distribution(using potassium antimonite precipitate method), ABA contents and protein synthesis patterns in pollinated stigma and ovaries of cucumber Xintaimici were investigated under 45 Cand 28 C after exogenous Ca2+ and ABA treatment. Under 45 C, Exogenous Ca2+ reduce the intercellular Ca2+ of cucumber stigma andovaries after pollination, while the exogenous ABA have indistinctive effect. Endogenous ABA contents in stigma and ovaries reduced significantly linked w更多还原