High-level expression and intracellular transport of foreign proteins in transformed rice.

通过在细菌β-葡萄糖苷酸酶(GUS)编码序列中引入内含子，我们可以在CAMV 35S启动子的控制下将GUS在转化的水稻细胞中的表达水平提高约100倍。相比之下，这个嵌合的35S-内含子：GU的表达水平。在转化烟草细胞中，S基因与35S：GUS基因的同源性较高。在转化35S-内含子：GUS的水稻细胞中，我们只能观察到得到内含子剪接的GUS mRNAs。另一方面，在转化的烟草细胞中，约有一半的转录本含有未剪接的内含子，这表明该内含子的剪接引入到GUS编码序列中在烟草细胞中不能有效剪接，尽管该内含子来自双子叶植物。尽管剪接效率不同，但内含子在水稻和烟草细胞中的剪接位置是相同的。除了剪接效率的差异外，Lev…转化水稻和烟草细胞中GUS-mRNAs的EL显著高于转化烟草细胞。我们利用甘薯线粒体F_1-ATPase三角洲亚基前体的N-末端前序列，检测了转化水稻和烟草细胞中GUS蛋白向线粒体的转运。前F_1三角洲含有由45个氨基酸残基组成的前序列，其中N-端23个氨基酸可形成两亲性的α-螺旋结构。在水稻和烟草细胞中，含有来自前F_1三角洲的N端46个氨基酸和来自前F_1三角洲的N端23个氨基酸的前F_1 Delta-GUS不支持GUS蛋白向线粒体的转运。只有当来自前F_1 Delta的N端73个氨基酸残基与水稻、烟草和酵母细胞中的GUS编码序列融合时，GUS蛋白才能向线粒体转运。这些结果表明，水稻细胞中蛋白质向线粒体运输的机制与其他器官中的类似。较少的GUS蛋白向线粒体运输

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 … More 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