摘要
植物原生质体是除去细胞壁后仍具有活力的裸露单细胞,其在植物RNA病毒的复制机理研究方面具有广泛的应用。为了获得高产、高活力的烟草原生质体,以烟草(Nicotiana tabacum Linn.)品种K326为材料,对原生质体制备体系中的不同细胞壁酶解液成分、酶解时间、酶解渗透压稳定剂含量、离心时间以及离心转速等因素进行优化。结果表明:在含1%纤维素酶,0.5%离析酶,0.6mol·L-1甘露醇的酶解液中,26℃慢速震荡(40r·min-1)酶解4h,700r·min-1离心3min,得到产量及活力较高的原生质体,可直接用于植物病毒的接种试验;利用聚乙二醇(PEG)融合法向获得的高活力原生质体中接种马铃薯Y病毒N株系(Potato virus Y necrosis strain,PVYN),经培养后提取总RNA,通过PVYN特异性引物经RT-PCR扩增后检测条带明显,表明PVYN成功导入烟草原生质体并自我复制。
Plant protoplasts a re naked living cells without cell wall, which are widely applied on the study of RNA virus replication mechanisms. In order to obtain tobacco protoplasts with high yield and vitality, condition optimization was performed in this study using tobacco variety K326 in the isolation and purification of tobacco protoplasts. Conditions such as combination of cellulose, concentration of mannitol, centrifugal speed and time were investigated. The results showed that the optimum conditions for the highest yield and vitality of protoplasts were as follows: enzyme combinations containing 1% cellulose, 0.5% pectinase, and 0.6mol·L-1mannitol, slow shocks(40r·min-1)at 26℃ and enzymatic hydrolysis time 6h, 700r·min-1centrifugal 3min. The isolated tobacco protoplasts were inoculated with Potato virus Y necrosis strain(PVYN) by using polyethylene glycol(PEG) method, subsequently incubated for 18-24h. Total RNA was extracted and amplified using RT-PCR by specific primers for PVYN. The results show that the PVYNwas successfully inoculated and replicated in tobacco K326 protoplasts.
出处
《沈阳农业大学学报》
CAS
CSCD
北大核心
2015年第1期26-30,共5页
Journal of Shenyang Agricultural University
基金
中国烟草总公司科技重点项目(中烟办[2010]221号)
高等学校博士学科点专项科研基金项目(20132103120014)
国家自然科学基金项目(31401710)
