Enhanced transgene expression in sugarcane by co-expression of virus-encoded RNA silencing suppressors.
Enhanced transgene expression in sugarcane by co-expression of virus-encoded RNA silencing suppressors.
复制标题
DOI：
10.1371/journal.pone.0066046
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Mirkov TE
中科院分区：
文献类型：
--
作者：
Gao SJ;Damaj MB;Park JW;Beyene G;Buenrostro-Nava MT;Molina J;Wang X;Ciomperlik JJ;Manabayeva SA;Alvarado VY;Rathore KS;Scholthof HB;Mirkov TE
转录后基因沉默在多倍体物种中被普遍观察到，并且常常是通过生物技术改良植物的主要限制因素。对5种植物病毒RNA沉默抑制剂在甘蔗中的作用进行了评价，研究了它们对甘蔗中增强的黄色荧光蛋白(EYFP)或β葡萄糖苷酸酶(GUS)报告基因表达的抑制作用。甘蔗是一种主要的糖类和生物量多倍体。这些抑制子的功能首先在烟草和洋葱表皮细胞中得到验证，然后通过在甘蔗幼叶切段和原生质体中的瞬时表达来验证。在共表达抑制子的幼叶片段中，EYFP在DNA导入后48-96h达到最大值，并且与没有抑制子的幼叶相比，EYFP在更长的时间内保持其表达高峰。其中番茄丛矮病毒编码的P19和大麦条纹花叶病毒编码的γb抑制效果最好。P19和γb的共表达使EYFP在幼叶中的表达分别增加了4.6倍和3.6倍，在原生质体中的GUS活性分别是无抑制子时的2.3倍和2.4倍。在转基因甘蔗中，GUS和P19抑制子的共表达表现出最高的GUS积累水平，平均是单独表达GUS时的2.7倍以上，并且没有不良的表型效应。以甘蔗幼叶切段和原生质体为基础建立的两种瞬时表达分析方法，经转基因稳定表达证实，为验证RNA沉默抑制子的有效性提供了一个快速、通用的系统，在甘蔗转基因表达的增强和稳定方面具有重要价值。
Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.
登录
查看更多内容
影响因子：
4.9
作者：
Chen, Songbiao;Tao, Lizen;Wang, Guo-Liang
通讯作者：
Wang, Guo-Liang
DOI：
10.1073/pnas.95.22.13079
发表时间：
1998-10-27
影响因子：
11.1
作者：
Anandalakshmi, R;Pruss, GJ;Vance, VB
通讯作者：
Vance, VB
影响因子：
6.2
作者：
Chiera, Joseph M.;Bouchard, Robert A.;Finer, John J.
通讯作者：
Finer, John J.
影响因子：
3
作者：
Chiera, Joseph M.;Lindbo, John A.;Finer, John J.
通讯作者：
Finer, John J.
影响因子：
6.2
作者：
CHOWDHURY, MKU;VASIL, IK
通讯作者：
VASIL, IK