月季花瓣中农杆菌介导的基因瞬时表达体系的优化及其在RNAi中的应用  

Optimization of Agrobacterium- mediated Transient Gene Expression System and Its Utilization in RNAi Based Gene Silencing of Rose(Rosa hybrida) Petals

作　　者：王磊[1] 陈雯[1] 刘娅[1] 杨若韵 阴霞 马男[1] 高俊平[1] 

机构地区：[1]中国农业大学观赏园艺与园林系,北京100193

出　　处：《农业生物技术学报》2014年第2期133-140,共8页Journal of Agricultural Biotechnology

基　　金：农业部948重点项目(No.2011-G17)

摘　　要：在植物中通过农杆菌介导的基因瞬时表达和基于RNAi的基因沉默可便捷高效地研究基因功能。本研究采用农杆菌(Agrobacterium tumefaciens)注射侵染法,利用GUS作为报告基因,探讨了月季(Rosa hybrida)品种、农杆菌菌株、开花级别、花瓣位置、农杆菌侵染浓度和侵染液成分等因素对月季花瓣中基因瞬时表达效果的影响。结果表明,月季品种蜜糖3级切花的中层花瓣中瞬时表达效果最佳;农杆菌菌株GV3101介导的瞬时表达效果最好,侵染浓度以OD600=0.9为宜,添加10 mmol/L MgCl2及10 mmol/L MES的侵染液效果优于重蒸水。同时,利用上述优化瞬时表达体系进行RNAi沉默,成功抑制了外源报告基因GUS和内源基因RhSAG的表达。相比对照,RhSAG沉默花瓣的萎蔫速度较慢,说明衰老进程得到延缓。该体系的优化为月季花瓣中基因功能的鉴定提供了有效工具。The gene functional identification in roses is limited by the lack of efficient stable transformation protocols. As an alternative, Agrobacterium-mediated transient gene expression and RNAi-based gene silencing can be used conveniently and effectively in plants. To optimize a transient gene expression system in rose petals, we investigated the effects of rose cultivars, flowering stages, petal positions, Agrobacterium strains, bacterial density and infiltration solution on transient gene expression in rose（Rosa hybrida） petals. The agro-infiltration method was used and GUS was selected as a marker gene in this study. We found that the cultivar Honey could have strongest GUS expression among all the 5 cultivars tested, while the other 4 cultivars Hollywood, Samantha, Honeymoon and Xiushanhong showed weak or no GUS expression. Petals of the middle whirl at flowering stage 3 displayed the highest expression level of GUS in Honey, suggesting that vigorous and fully developed petals were more suitable for transient gene expression. Compared to Agrobacterium strains of C58C1 and EHA105, GV3101 was a more effective laboratory strain and the optimal bacterial density was OD600=0.9. The infiltration solution is better to be supplemented with 10 mmol/L MgCl2 and 10 mmol/L 2-（N-Morpholino） ethanesulfonic acid （MES）. The optimized transient system could result in around 100% strong GUS expression. By using the optimized transient expression system, we transiently expressed ihpRNA constructs to silence the reporter gene GUS and RhSAG. Compared to control, GUS-silencing showed less strongly stained petals and a lower average staining density, while RhSAG-silenced petals wilted more slowly, indicating senescence delay. In addition, both silencing treatments led to decreased gene transcripts. Together, the results suggested the system could be used in gene silencing and provide an effective tool for gene function analysis in rose petals.

关 键 词：月季 花瓣 农杆菌 瞬时表达 RNAI 

分 类 号：S511[农业科学—作物学]