|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1997 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1996 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1995 : ¥1,100,000 (Direct Cost : ¥1,100,000)
The expression of 1724rolB gene (rolB gene of pRil724 harbored in Agrobacterium rhizogenes strain MAFF301724 which has been isolated in Japan) regulated by auxin compounds was investigated. Firstly, by the determination of nucleotide sequence of the right side of T-DNA,a right border and a T-DNA transfer stimulator sequence (TSS) were identified. A new open reading frame (ORF) X was also identified. Secondly, the detailed status of the transcription of genes on pRi1724 core-T-DNA was investigated by northern blotting and a reverse transcription-polymerase chain reaction (RT-PCR) analysis. The transcripts according to each ORF were detected.
By addition of NAA (naphthaleneacetic acid, a compound of auxin), the activation of transcription of 1724rolB gene was initiated within 6 hr and reached maximum within 12 hr. For identifying the region response to auxin, the promoter region of 1724rolB gene (from 1 to 1202 bp upstream of ATG codon) was isolated and integrated into the upstream of bet
a-glucuronidase (GUS) gene. Tobacco hairy roots containing the construct were obtained and their GUS activities were measured by fluorescent method. The promoter region showed the GUS activity which had been one-tenth less than that of CaMV 35S promoter. In addition, the expression of 1724rolB promoter was observed in the epidermis and root hairs of the hairy roots by histochemical GUS staining method. A set of unidirectional deletion from the upstream of the 1724rolB promoter combined with GUS gene was constructed. Tobacco hairy roots containing each construct were obtained for estimating GUS activity by fluorescent method. GUS activity was decreased between 986 and 678 bp and the auxin response was lost between 678 and 540 bp, suggesting that a region of activation of transcription exists between 986 and 678 bp and a region response to auxin exists between 678 and 540 bp, respectively.
By contrast, a cis-element (ACTTTA) response to auxin in rolB promoter of pRiA4 has been identified by the other group. At present, for screening of trans-elements bound to the cis-element from a tobacco cDNA library, yeast one-hybrid system using a homologous sequence in pRi1724 including the cis-element has been in progress. Less