The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

中文翻译：

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富含GC的编码序列减少了类似转座子的小RNA介导的转基因沉默。

植物对转基因沉默的分子基础知之甚少；因此，我们评估了几种转基因设计参数，以减少可遗传的转基因沉默。用编码微藻类多不饱和脂肪酸（PUFA）合酶的转基因对拟南芥植物进行的分析显示，小RNA（sRNA）介导的沉默，与重复调控元件的结合使用，导致油菜偏向PUFA合酶转基因的转座子样沉默。 。多样化的调控序列和使用具有较高GC含量的天然微藻编码序列（CDS）可改善转基因表达，并通过减少sRNA的积累和DNA甲基化而产生显着的跨代稳定性。在玉米中分别表达来自苏云金芽孢杆菌（Bt）的三种晶体（Cry）蛋白的转基因玉米的进一步实验测试了使用不同密码子偏倚表对CDS编码的影响。具有较高GC含量的转基因表现出增加的转录本和蛋白质积累。这些结果表明，转基因CDS的序列组成可以直接影响沉默，为提高转基因表达水平和降低遗传损失的遗传风险提供了设计策略。