Cloning of genes preferentially expressed in early stage of parthenocarpic and pollinated fruit and stabilization of fruit set

通过荧光显微镜观察茄子花授粉后花粉管伸长，顶端在授粉后1.5~2天内到达子房胚珠。为此，在开花去势后，设置了授粉、未授粉和未授粉+生长素处理三个实验条件。培养2~3d后，提取每个样本的RNA。对于授粉后2天的表达，通过已授粉的果实和未授粉的果实之间的差减，得到了消减的cDNA文库。我们随机挑选了384个克隆，并将它们斑点在细胞膜上，用于大分子阵列分析。筛选出32个授粉果实比未授粉果实高表达基因的候选克隆作为探针进行Northernb杂交分析。在这32个克隆中有11个显示了…此外，授粉果实的杂交信号比未授粉果实的杂交信号更强。与P450相似的3个基因、未知蛋白和假想蛋白在授粉果实和生长素处理的果实中均在开花后2~4d特异表达，然后迅速减少。另一方面，在授粉后3d的表达中，有10个克隆优先表达，包括组蛋白、甘油三酯、多酚氧化酶、细胞壁结构蛋白、细胞分裂相关蛋白、分子伴侣。然而，几乎所有的mRNAs在3d后仍有明显的表达。在甜椒果实中，7个EST基因在授粉后2天优先在授粉果实中表达，这7个基因与茄子果实相同。具有开花调控功能的胚体花2在授粉过程中显著表达。较少

The purpose of this project is to make clear the genes preferentially relating to parthenocarpy and fruit setting for its stabilization.It was obserbed using fluorescence microscopy that pollen tube elongated after pollination of eggplant flower and the tip reached the ovules in ovary within 1.5 to 2 days after pollination. Therefore, three experimental conditions of pollination, unpollination, and unpollination + auxin treatments were set after emasculation on the flower opening. And RNAs from each sample were extracted after 2 or 3 days growth. Regarding the expression on 2 days after pollination, subtracted cDNA library was prepared by the subtraction between pollinated and unpollinated fruit. We picked up randumly 384 cDNA clones and spotted them on the membrane for macroarray assay. Thirty two candidates of cDNA clones for genes expressed higher in pollinated fruit than unpollinated one were selected and used as probes for Nothern blot analysis. Eleven of these 32 cDNA clones show … More ed that hybridization signals with mRNA from pollinated fruit were stronger ttian that from unpollinated fruit. mRKAs of three genes having similarity to P450, Unknown protein, and Hypothetical protein were specifically expressed in both pollinated and auxin treated fruit from 2 to 4 days after anthesis and then reduced rapidly. On the other hand, regarding the expression on 3 days after pollination, there were 10 cDNA clones expressed preferentially, in which histone, carleticulin, polyphenol oxidase, cell wall structural protein, cell division relating protein, molecular chaperon were contained. However, almost all mRNAs continued to be expressed clearly ever after 3 days. Thus, it was suggested that the 3 genes expressed on 2 days related to the initiation of fruit set.In sweet pepper fruit, also, 7 EST genes preferentially expressed in pollinated fruit on 2 days after pollination were isolated using the same way to eggplant fruit. Embryomic flower 2, which functions the regulation of flowering, was prominently expressed by pollination. Less