CONTROL OF SELFINCOMPATIBILITY IN JAPANESE PEAR USING GENE MODIFICATION

具有RNase活性的S等位基因相关花柱糖蛋白（S-RNase）是雌蕊S基因的产物。S-RNase的表达在花发育过程中增加，在开花前达到最大值。为了确定S-RNase启动子上的ds调控元件，我们分析了日本梨S-RNase基因组的5个侧翼区域。从日本梨‘nijisisseiki’（S_2S_4）、‘Hohsui’（S_3S_5）和‘Kohsui’（S_4S_5）三个基因组文库中分别分离出S_2-、S_3-和S_5-RNases的基因组dna，并进行了测定。对S_2-、S_3-、S_4-和S_5-RNases基因组中三个5个侧翼区域的比较表明，在假定的TATA盒的上游区域存在一个高度相似的约200 bp的区域（框1，从-120到-325），这表明该区域存在可能的顺式调控元件。框1上游的区域在4个日本梨s - rnase基因中是异质的，尽管框1上游的区域（框2，从-326到-507）在S_3和S_5-RNase基因之间检测到180…更多bp的同源区域。为了鉴定日本梨S-RNase启动子上的cz调控元件，我们分析了9个与β-葡萄糖醛酸酶（GUS）基因融合的S_3-RNase启动子序列缺失的转基因烟草植株的GUS活性。在携带-747 ~ -310启动子的转基因植株雌蕊中定量检测到GUS活性。GUS活性主要集中在柱头分泌区和雌蕊传递组织，其他组织不存在；花粉、花药、花瓣、萼片、叶和根。从开花前4天开始，雌蕊中GUS活性逐渐增加。缺失-255或更短启动子的转基因雌蕊没有GUS活性，说明S_3-RNase启动子区-310 ~ -255之间存在雌蕊特异性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活性急剧下降，这表明该区域存在雌蕊中高水平表达的增强子样元件。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