| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥2,800,000 (Direct Cost: ¥2,800,000)
|
|---|
| Research Abstract |
In the cross Nicotiana suaveolens x N.tabacum, Yamada et al. (2001) reported that, using suspension cultured cells, hybrid lethality is expressed at 28℃, but suppressed at 36℃ and cell death during hybrid lethality in this hybrid is apoptotic and performed under genetic control. Masuda et al. (2003) reported that the point of no return, when the factor(s) controlling hybrid lethality are expressed, is 3 h after exposure to 28℃ and after the point of no return de novo transcription and translation are not required. In the present study, we attempted to isolate the genes expressing during hybrid lethality using the cDNA subtraction method. Poly(A)+RNA was isolated from a lethal hybrid line (LH line) and a surviving hybrid line (SH line) exposed to 28℃ for 5 h. Subtractive hybridization was performed using a PCR-select cDNA subtraction kit (Clontech) following the manufacturer's instructions. The subtracted cDNAs were cloned into a pGEM-T-easy vector (Promega) and secondary screening was performed using a PCR-select differential screening kit (Clontech). Eighteen positive clones were obtained and sequenced. A search using the BLASTX program identified similar sequences deposited within the GenBank database. To investigate the expression of genes specific to hybrid lethality, the real-time quantitative PCR was performed using an iQ SYBR green super mix (BioRad) on cDNA Obtained from the LH and SH lines exposed to 28℃ for 0,3,and 6 h. As a result, senescence (LH16,LH84), hypersensitive response (LH25), and auxin-induced protein (LH3)-related genes were expressed specifically in the LH line. This data suggests that hybrid lethality may possess similar mechanisms in the commitment or execution phases with senescence or hypersensitive response.
|
