Post-transcriptional silencing of pectin methylesterase gene in transgenic tomato fruits results from impaired pre-mRNA processing
Post-transcriptional silencing of pectin methylesterase gene in transgenic tomato fruits results from impaired pre-mRNA processing
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DOI：
10.1046/j.1365-313x.1998.00157.x
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发表时间：
1998-06-01
期刊：
影响因子：
7.2
通讯作者：
Handa, AK
中科院分区：
文献类型：
--
作者：
Mishra, KK;Handa, AK
在CaMV 35 S启动子控制下表达截短的果实果胶甲酯酶（PME）正义转基因的转基因番茄果实显示内源果实PME和正义转基因的发育调节共沉默。这种共沉默与果实PME基因表达的开始有关。PME蛋白和mRNA的检测水平不存在成熟的转基因水果。然而，在转基因果实中存在与转基因的3'末端杂交的几种较小的转基因RNA种类，表明有义转基因转录物的降解。从野生型和转基因水果的核RNA的RT-PCR分析，引发与寡核苷酸（dT），显示剪接和未剪接的转录本的水果PME和转基因的存在下，表明基因沉默的转录后调节。然而，在PCR扩增中，使用随机引物的cDNA在转基因果实的核中获得未剪接的果实PME转录物的积累。两者合计，这些结果表明，果实PME正义转基因的异位表达干扰内源性PME前mRNA的加工。
Transgenic tomato fruit expressing a truncated fruit pectin methylesterase (PME) sense transgene under the control of CaMV 35S promoter show developmentally regulated co-silencing of the endogenous fruit PME and sense transgene. This co-silencing is associated with the onset of fruit PME gene expression. Detectable levels of PME protein and mRNA were not present in ripening transgenic fruits. However, several smaller transgene RNA species hybridizing to the 3' end of the transgene are present in transgenic fruits, indicating degradation of sense transgene transcript. RT-PCR analyses of nuclear RNA from wild-type and transgenic fruits, primed with oligo(dT), show the presence of spliced and unspliced transcripts for the fruit PME and the transgene, indicating a post-transcriptional regulation of gene silencing. However, an accumulation of unspliced fruit PME transcript was obtained in the nuclei of transgenic fruit, using the randomly primed cDNAs in PCR amplification. Taken together, these results suggest that the ectopic expression of the fruit PME sense transgene interferes with processing of the endogenous PME pre-mRNA.