钙在蓝猪耳双受精过程中作用的研究
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摘要
钙在被子植物双受精过程的作用一直是植物学家的兴趣热点。早期有关钙对植物有性生殖影响的研究主要集中在研究钙对花粉萌发和花粉管生长的作用方面。与花粉萌发和花粉管生长过程中的作用的研究相比，对雌蕊组织中，尤其是胚囊中的钙的作用的研究十分有限。到目前为止，对植物胚囊中钙的研究也绝大多数局限于总钙和膜钙的的测定，而对于被子植物胚囊内胞质游离钙的研究则几乎是空白。这方面的研究之所以比较滞后，一方面是由于大多数被子植物胚囊深藏于子房内，为处理和观察带来很大困难。同时研究胚囊胞质游离钙的技术手段也有很大的局限。因此，材料本身的结构限制和研究手段的限制使得植物双受精过程中胚囊内胞质游离钙的研究相比之下显得落后。
针对这种现状，本工作选用具有裸露胚囊的玄参科的蓝猪耳植物为研究材料，研究Ca~(2+)与受精过程中相关的细胞学事件的关系，探索[Ca~(2+)]_(cyt)在被子植物双受精过程中的作用。工作之一首先是检测受精过程中雌性生殖单位中发生的显著的细胞学变化，以此作为受精与否的标志；然后经过外源导入钙(A23187诱导)研究[Ca~(2+)]_(cyt)在受精过程中的这一细胞学事件发生中所起的作用。另一相关的工作是构建转YC2.1基因的蓝猪耳植株(YC2.1是钙的荧光指示剂)，然后对不同发育时期的蓝猪耳胚囊中[Ca~(2+)]_(cyt)的动态变化进行研究。
通过荧光染色技术，我们首先观察了自然受精过程中胚囊内雌性生殖单位细胞壁的变化。结果表明，开花前蓝猪耳的卵细胞的胞壁是完整的；开花时胚囊已发育成熟，此时卵细胞在合点端部分区域是裸的；受精后(大约授粉后20小时)卵细胞的胞壁恢复完整。表明细胞壁的合成是受精后早期发生的细胞学事件。在此基础上，我们分离了胚囊细胞的原生质体。通过外源导入钙离子检查钙能否引发类似受精过程发生的细胞学事件。结果显示：外源钙的流入导致卵细胞内发生了与钙有关的细胞学事件，但不同状态的雌性生殖单位的细胞原生质体对外源钙的流入产生不尽相同的反应。胚囊已发育成熟(柱头已张开)时期的未受精的卵细胞原生质体，钙离子载体A23187处理后引发了卵细胞壁的形成，类似于发生在自然受精过程中的现象，而在对开花前分离的卵细胞原生质体以及受精后(授粉后20小时)分离的卵细胞原生质体作相同的处理后则未发现细胞壁的再生；同作为双受精参与者之一的中央细胞经过处理后，无论受精前还是受精后(授粉后20小时)均未发现细胞壁的再生。这些结果表明，外源钙流入导致的胞质Ca~(2+)浓度的升高，有促进卵细胞细胞壁快速形成的作用；而且，卵细胞对Ca~(2+)信号的反应与发育状态有关，可能仅成熟的未受精的卵细胞处于一种特殊的“感受态”，能对Ca~(2+)信号发生反应而快速形成细胞壁。
转基因蓝猪耳植株构建工作已取得阶段性结果。从质粒扩增、农杆菌转化、侵染一直到叶盘法转化蓝猪耳，经过多次筛选，得到抗性苗，目前正在筛选表达YC2.1荧光指示剂的转基因苗。
Functions of Calcium during fertilization of angiosperm is one of research focuses for botanists. Prior studies of calcium on sexual reproduction of angiosperm mainly covered these about pollen germination and tube growth processes; there are, however, few works on pistil tissue. So far, achieved studies on calcium in embryo sac of angiosperm mostly located in memebrane-binding calcium and total calcium but little in free cytoplasmic calcium ([Ca2+]cyt ). Obstacles to progress are : (1) the double fertilization of most angiosperms occurs internally, deep within ovary tissues, as a result it is very difficult to investigate the truth. (2) There are few ideal techniques for mearsuring [Ca2+]cyt. In conclusion, both limitation delay studies of [Ca2+]cyt in embryo sac during fertilization.
The thesis work is to investigate the connection of [Ca2+]cyt and fertilization by avoiding drawbacks noted above, say, employing an ideal experimental plant, Torenia fournieri , with a naked embryo sac that protrudes from the micropyle. Methods to be applied include extracellular Ca2+ influx treatment and intracellular Ca2+ measure by transgenical expression of Ca2+ fluorescence indicator during fertilization. The former is first to obtain protoplasts of female germination unit in different development stages, then Ca2+ lonophore treatment; the latter is to express transgenically the Ca2+-sensing cameleon protein in Torenia fournieri, which allows long term measuring stimulus-induced cytoplasmic Ca2+ changes.
First we employed C.W.M2R, a fluorescent fabric brightener, to investigate changes of wall of female germ unit before and after fertilization. Before anthesis wall of egg apparatus is complete; most part of egg wall at the chalazal end disappears at the day of anthesis; after frtilization (20h after pollinating) egg wall becomes intact. The results suggest that rebuilding of wall is one of the early cytological events during fertilization. Based on these observations, we isolated protoplasts from female germ unit and performed extracellular Ca2+ influx experiment in order to check whether the cytological event similar to what happens during fertilization will be induced by extracellular Ca2+ influx. The results of extracellular Ca2+ treatment revealed that influx of extracellular Ca2+ induced some cellular event and protoplasts of female germ unit in different development stages respond to the treatment differently. Ca2+ ionophore treatment induced occurrence of new cellulosic wall around protoplasts of unfe
rtilized egg cells from mature embryo sac , which just does occur during fertilization. New wall in protoplasts from fertilized egg cells or from egg cells before anthesis, however, weren't detected, as well as protoplasts of central cell. These results indicated that the increase of cytoplasmic Ca2+ from extracellular Ca2+ influx contributes to the rapid establishment of cellulosic cell wall. Moreover, these results also suggest that only mature unfertilized egg cells, which are at a "special" state, are able to respond to Ca2+ signal to establish new wall rapidly.
As for construction of transgenic Torenia fournieri, some foundermantal works have been done including plasmid amplification, Agrobactarium transformation, leaf disk transformation- regeneration, etc.. The work for screening transgenic plant which expresses YC2.1 is in progress.
针对这种现状，本工作选用具有裸露胚囊的玄参科的蓝猪耳植物为研究材料，研究Ca~(2+)与受精过程中相关的细胞学事件的关系，探索[Ca~(2+)]_(cyt)在被子植物双受精过程中的作用。工作之一首先是检测受精过程中雌性生殖单位中发生的显著的细胞学变化，以此作为受精与否的标志；然后经过外源导入钙(A23187诱导)研究[Ca~(2+)]_(cyt)在受精过程中的这一细胞学事件发生中所起的作用。另一相关的工作是构建转YC2.1基因的蓝猪耳植株(YC2.1是钙的荧光指示剂)，然后对不同发育时期的蓝猪耳胚囊中[Ca~(2+)]_(cyt)的动态变化进行研究。
通过荧光染色技术，我们首先观察了自然受精过程中胚囊内雌性生殖单位细胞壁的变化。结果表明，开花前蓝猪耳的卵细胞的胞壁是完整的；开花时胚囊已发育成熟，此时卵细胞在合点端部分区域是裸的；受精后(大约授粉后20小时)卵细胞的胞壁恢复完整。表明细胞壁的合成是受精后早期发生的细胞学事件。在此基础上，我们分离了胚囊细胞的原生质体。通过外源导入钙离子检查钙能否引发类似受精过程发生的细胞学事件。结果显示：外源钙的流入导致卵细胞内发生了与钙有关的细胞学事件，但不同状态的雌性生殖单位的细胞原生质体对外源钙的流入产生不尽相同的反应。胚囊已发育成熟(柱头已张开)时期的未受精的卵细胞原生质体，钙离子载体A23187处理后引发了卵细胞壁的形成，类似于发生在自然受精过程中的现象，而在对开花前分离的卵细胞原生质体以及受精后(授粉后20小时)分离的卵细胞原生质体作相同的处理后则未发现细胞壁的再生；同作为双受精参与者之一的中央细胞经过处理后，无论受精前还是受精后(授粉后20小时)均未发现细胞壁的再生。这些结果表明，外源钙流入导致的胞质Ca~(2+)浓度的升高，有促进卵细胞细胞壁快速形成的作用；而且，卵细胞对Ca~(2+)信号的反应与发育状态有关，可能仅成熟的未受精的卵细胞处于一种特殊的“感受态”，能对Ca~(2+)信号发生反应而快速形成细胞壁。
转基因蓝猪耳植株构建工作已取得阶段性结果。从质粒扩增、农杆菌转化、侵染一直到叶盘法转化蓝猪耳，经过多次筛选，得到抗性苗，目前正在筛选表达YC2.1荧光指示剂的转基因苗。
Functions of Calcium during fertilization of angiosperm is one of research focuses for botanists. Prior studies of calcium on sexual reproduction of angiosperm mainly covered these about pollen germination and tube growth processes; there are, however, few works on pistil tissue. So far, achieved studies on calcium in embryo sac of angiosperm mostly located in memebrane-binding calcium and total calcium but little in free cytoplasmic calcium ([Ca2+]cyt ). Obstacles to progress are : (1) the double fertilization of most angiosperms occurs internally, deep within ovary tissues, as a result it is very difficult to investigate the truth. (2) There are few ideal techniques for mearsuring [Ca2+]cyt. In conclusion, both limitation delay studies of [Ca2+]cyt in embryo sac during fertilization.
The thesis work is to investigate the connection of [Ca2+]cyt and fertilization by avoiding drawbacks noted above, say, employing an ideal experimental plant, Torenia fournieri , with a naked embryo sac that protrudes from the micropyle. Methods to be applied include extracellular Ca2+ influx treatment and intracellular Ca2+ measure by transgenical expression of Ca2+ fluorescence indicator during fertilization. The former is first to obtain protoplasts of female germination unit in different development stages, then Ca2+ lonophore treatment; the latter is to express transgenically the Ca2+-sensing cameleon protein in Torenia fournieri, which allows long term measuring stimulus-induced cytoplasmic Ca2+ changes.
First we employed C.W.M2R, a fluorescent fabric brightener, to investigate changes of wall of female germ unit before and after fertilization. Before anthesis wall of egg apparatus is complete; most part of egg wall at the chalazal end disappears at the day of anthesis; after frtilization (20h after pollinating) egg wall becomes intact. The results suggest that rebuilding of wall is one of the early cytological events during fertilization. Based on these observations, we isolated protoplasts from female germ unit and performed extracellular Ca2+ influx experiment in order to check whether the cytological event similar to what happens during fertilization will be induced by extracellular Ca2+ influx. The results of extracellular Ca2+ treatment revealed that influx of extracellular Ca2+ induced some cellular event and protoplasts of female germ unit in different development stages respond to the treatment differently. Ca2+ ionophore treatment induced occurrence of new cellulosic wall around protoplasts of unfe
rtilized egg cells from mature embryo sac , which just does occur during fertilization. New wall in protoplasts from fertilized egg cells or from egg cells before anthesis, however, weren't detected, as well as protoplasts of central cell. These results indicated that the increase of cytoplasmic Ca2+ from extracellular Ca2+ influx contributes to the rapid establishment of cellulosic cell wall. Moreover, these results also suggest that only mature unfertilized egg cells, which are at a "special" state, are able to respond to Ca2+ signal to establish new wall rapidly.
As for construction of transgenic Torenia fournieri, some foundermantal works have been done including plasmid amplification, Agrobactarium transformation, leaf disk transformation- regeneration, etc.. The work for screening transgenic plant which expresses YC2.1 is in progress.
引文
胡适宜等．被子植物受精生物学． 2002：4-108
孙大业等．细胞信号转导．北京：科学出版社，1998：171-285
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