CONTROL OF SELFINCOMPATIBILITY IN JAPANESE PEAR USING GENE MODIFICATION

具有核糖核酸酶活性的S等位基因相关的花柱糖蛋白(简称S核糖酶)已被鉴定为雌蕊中S基因产物。S核糖核酸酶在花发育过程中表达增强，在开花前达到最大值。为了鉴定S核糖核酸酶启动子上的双链调控元件，分析了日本梨S核糖核酸酶基因组的5个侧翼区域。分别从‘日本梨’(S_2S_4)、‘河水’(S_3S_5)和‘高穗’(S_4S_5)的3个基因组文库中提取了日本梨S_2、S_3和S_5-核糖核酸酶的基因组DNA，并进行了测定。对S_2-、S_3-、S_4-和S_5-核糖核酸酶基因组的5个侧翼区的比较表明，在TATA盒的上游存在一个约200bp的高度相似的区域(盒1，从-120到-325)，这表明该区域存在可能的顺式调控元件。在4个日本梨S核糖核酸酶中，盒1的上游区域是异质性的，尽管另外还有180个…在S_3和S_5-核糖核酸酶基因的上游发现了更多的核苷酸同源区域(盒2，从-326到-507)。为了鉴定日本梨S-核糖核酸酶启动子上的CZS调控元件，我们分析了9个序列缺失的S_3-核糖核酸酶启动子与β-葡萄糖苷酸酶(GUS)基因融合的烟草植株中的GUS活性。在携带-747~-310缺失启动子的转基因植株的雌蕊中检测到GUS活性。GUS活性定位于柱头分泌区，传递组织定位于雌蕊，但不存在于花粉、花药、花瓣、萼片、叶片和根中，雌蕊中GUS活性从开花前4d开始逐渐升高。带有-255或更短启动子的转基因雌蕊均未表现出GUS活性，表明S_3核糖核酸酶启动子区域-310~-255之间存在调控雌蕊特异表达的关键顺式调控元件。此外，从-552到-356的缺失导致转基因雌蕊中GUS活性的急剧下降，这表明该区域内存在促进雌蕊高水平表达的增强子样元件。较少

S-allele-associated stylar glycoproteins with RNase activity (termed S-RNases) have been identified as S gene products in pistil. The expression of S-RNase is increased during flower development and maximized before anthesis. To identify the ds-regulatory elements on the S-RNase promoter, we analyzed the 5 flanking regions in the genomic of Japanese pear S-RNases.Genomic DNAs of S_2-, S_3-, and S_5-RNases of Japanese pears, respectively, were isolated from three genomic libraries of 'Nijisseiki' (S_2S_4), 'Hohsui' (S_3S_5) and 'Kohsui' (S_4S_5), and then determined. Comparison of three 5 flanking regions in the genomic of S_2-, S_3-, S_4- and S_5-RNases indicated that a highly similar region of approximately 200 bp (box 1, from -120 to -325) exists in the regions just upstream of the putative TATA boxes, which suggests the presence of may cis-regulatory elements in this region. The regions upstream of box 1 are heterogeneous among the four Japanese pear S-RNases although a further 180 … More bp homologous region (box 2, from -326 to -507) just upstream of the box 1 is detected between S_3 and S_5-RNase genes.To identify the czs-regulatory elements on the S-RNase promoter of Japanese pear, we analyzed GUS activities in transgenic tobacco plants carrying 9 sequential deletions of S_3-RNase promoter fused to the β-glucuronidase (GUS) genes. GUS activity was detected quantitatively in the pistils from the transgenic plants carrying the promoter deleted from -747 to -310. GUS activity was localized in the stigmatic secretory zone, and the transmitting tissue in the pistils but not in other tissues ; pollen, anthers, petals, sepals, leaves and roots.GUS activities in the pistils increased steadily from 4 days before flowering. No transgenic pistils with deletions carrying -255 or shorter promoters displayed GUS activity, indicating that the critical cis-regulatory elements for pistil specific expression were present between -310 and -255 of S_3-RNase promoter region. Furthermore, deletions from -552 to -356 led to the drastic decrease of GUS activity in the transgenic pistil, which assume that enhancer-like element for the high level of expression in pistils are present within this region. Less