KATAYAMA Hironori FACULTY OF AGRICULTURE, KOBE UNIVERSITY, ASSISTANT PROFESSOR, 農学部・付属農場, 助手 (50294202)
TAKASAKI Takeshi FACULTY OF AGRICULTURE, KOBE UNIVERSITY, ASSISTANT PROFESSOR, 農学部, 助手 (30314511)
|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 2001 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 2000 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1999 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1998 : ¥1,700,000 (Direct Cost : ¥1,700,000)
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
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