1991 Fiscal Year Final Research Report Summary
High-level expression and intracellular transport of foreign proteins in transformed rice.
| Project/Area Number |
02454061
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用生物化学・栄養化学
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| Research Institution | Nagoya University |
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Principal Investigator |
NAKAMURA Kenzo Nagoya University, School of Agriculture, Associate Professor, 農学部, 助教授 (80164292)
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| Co-Investigator(Kenkyū-buntansha) |
MORIKAMI Atsushi Nagoya University, School of Agriculture, Research Associate, 農学部, 助手 (10211608)
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| Project Period (FY) |
1990 – 1991
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| Keywords | Transgene expression / Efficiency of gene expression / Intron / Transformed rice cells / Transport of proteins to mitochondria |
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| Research Abstract |
By introducing an intron within the coding sequence of bacterial beta-glucuronidase(GUS), we could enhance the level of expression of GUS under the control of CAMV 35S promoter up to about 100-fold in transformed rice cells. By contrast, the level of expression of this chimeric 35S-Intron : GU. S gene was about the same as that of the 35S : GUS gene in transformed tobacco cells. In rice cells transformed with 35S-Intron : GUS, we could observe only GUS mRNAs that had received splicing of the intron. On th other hand, in transformed tobacco cells about half of the transcripts contained unspliced intron, indicating that the splicing of this intron introduced to the GUS-coding sequence is not efficiently spliced in tobacco cells albeit this intron was derived from the dicotyledonous plants. Despite of the difference in the efficiency of splicing, the site of splicing of the intron was identical in rice and tobacco cells. In addition to the difference in the efficiency of splicing, the lev
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el of GUS-mRNAs in transformed rice cells was significantly higher than that in transformed tobacco cells.
We examined the transport of GUS protein to mitochondria in transformed rice and tobacco cells using the N-terminal pre sequence of the precursor to the delta-subunit of sweet' potato mitochondrial F_1-ATPase. The pre-F_1delta contains the presequence composed of 45 amino acid residues of which the N-terminal 23 amino acids can form amphiphilical alpha-helix structure. The pre-F_1delta -GUS with the N-terminal 46 amino 'acids from the pre-F_1delta, as well as with the N-terminal 23 amino acids from the pre-F_1delta, did not support the transport of GUS protein to mitochondria both in rice and tobacco cells. The transport of GUS proteins to the mitochondria only occurred when the N-terminal 73 amino acid residues from the pre-F_1delta was fused to the GUS coding sequence in both rice, tobacco and yeast cells. These results indicated that the mechanism of transport of proteins to mitochondria in rice cells is similar to those operating in other organs. Less
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