CONTROL OF SELFINCOMPATIBILITY IN JAPANESE PEAR USING GENE MODIFICATION

利用基因修饰控制日本梨的自相容性

• 批准号: 10660025
• 负责人: NAKANISHI Tetsu
• 金额: $ 2.18万
• 依托单位: KOBE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660025/
• 关键词: SELF-INCOMPATIBILITY; JAPANESE PEAR; S ALLELE; S-RNase; PROMOTER; CIS REGULATORY ELEMENT; s-RNase; プロモーター解析; 遺伝子組換え

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

具有RNA酶活性的S等位基因相关的花柱糖蛋白（简称S-RNases）是S基因在雌蕊中的产物。S-RNase的表达在花发育过程中增加，并在开花前达到最大值。为了鉴定S-RNase启动子上的顺调控元件，我们对日本梨S-RNase基因组中的5个侧翼区进行了分析，分别从日本梨'Nijisseiki'（S_2S_4）、'Hohsui'（S_3S_5）和'Kohsui'（S_4S_5）基因组文库中提取了日本梨S_2-、S_3-和S_5-RNase基因组DNA，并进行了分析。对S_2-、S_3-、S_4-和S_5-RNases基因组中3个5-侧翼区的比较表明，在推定的TATA盒上游存在一个约200 bp的高度相似区（box 1，从-120到-325），这表明在该区域存在可能的顺式调控元件。框1的上游区域在四种日本梨S-RNase中是异质的，尽管另外180 ...更多信息 为了鉴定日本梨S-RNase启动子上的顺式调控元件，我们对转基因烟草植株进行了GUS活性分析，这些转基因烟草含有9个顺序缺失的S_3-RNase启动子和GUS基因。从-747 ~-310缺失启动子的转基因植株的雌蕊中定量检测GUS活性。GUS活性定位于雌蕊的柱头分泌区和传递组织中，但在花粉、花药、花瓣、萼片、叶片和根中不存在，雌蕊中的GUS活性从开花前4d开始逐渐增加。缺失-255或更短启动子的转基因雌蕊均未显示GUS活性，表明雌蕊特异表达的关键顺式调控元件位于S_3-RNase启动子区的-310 ~-255之间。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，这假定在雌蕊中高水平表达的增强子样元件存在于该区域内。少

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