Dow AgroSciences LLC., 9330 Zionsville Road, Indianapolis, IN, 46268, USA.
Delaware Biotechnology Institute, Department of Plant and Soil Sciences, University of Delaware, Newark, DE, 19716, USA.
Nat Plants. 2017 Nov;3(11):875-884. doi: 10.1038/s41477-017-0040-6. Epub 2017 Oct 30.
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.
转基因对沉默的敏感性的分子基础在植物中描述得很差；因此，我们评估了几种转基因设计参数，以期减少可遗传的转基因沉默。对编码微藻多不饱和脂肪酸（PUFA）合酶的转基因拟南芥植物的分析表明，小 RNA（sRNA）介导的沉默，加上使用重复的调控元件，导致油菜偏向的 PUFA 合酶转基因的转座子样沉默。多样化的调控序列并使用具有更高 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 和 DNA 甲基化的积累，导致显著的跨代稳定性。在玉米中分别用来自苏云金芽孢杆菌（Bt）的三种晶体（Cry）蛋白的转基因进行的进一步实验，测试了使用不同密码子偏好表对 CDS 重编码的影响。GC 含量较高的转基因表现出转录本和蛋白质积累增加。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转基因表达可遗传性丧失的风险提供了设计策略。
