烟草丛顶病毒编码的沉默抑制子初步鉴定  

Preliminary Identification of RNA Silencing Suppressors Encoded by Tobacco Bushy Top Virus

作　　者：张润[1,2] 王连春[1] 陈海如[1] 李凡[1] 

机构地区：[1]云南农业大学,农业生物多样性与病虫害控制教育部重点实验室,云南昆明650201 [2]云南农业大学农学与生物技术学院,云南昆明650201

出　　处：《云南农业大学学报（自然科学版）》2011年第6期749-754,共6页Journal of Yunnan Agricultural University:Natural Science

基　　金：国家自然科学基金(30660101)

摘　　要：烟草丛顶病毒(Tobacco bushy top virus,TBTV)为幽影病毒属成员,能在烟草上造成严重危害。TBTV基因组编码4个ORF,其中ORF3和ORF4几乎完全重叠,可能在TBTV的致病性中发挥重要作用。分别将TBTV的ORF3和ORF4与载体pGR107连接,构建植物表达载体pGR107-ORF3,pGR107-ORF4。经农杆菌与含外源GFP基因的pGREEN208共浸润转GFP基因的本氏烟16c叶片,2～5 d后(days post-inoculation,dpi)在紫外灯下观察发现浸润区有明显绿色荧光,其中pGR107-ORF4在12 dpi后浸润区绿色荧光区域扩大,推断ORF4编码产物可能具有局部抑制本氏烟16c对外源GFP基因进行沉默的功能;而pGR107-ORF3浸润区绿色荧光逐渐恢复红色,GFP基因发生沉默,说明ORF3编码产物不具有抑制基因沉默的功能。进一步利用real-time PCR定量检测发现,pGR107-ORF4浸润15 dpi的本氏烟叶片中GFP RNA的含量要比仅接种pGR107的对照高,比未接种的本氏烟16c稍低。推断是因为ORF4编码蛋白抑制了植物对外源GFP RNA产生的沉默,使GFP得以在本氏烟内表达。由此进一步证实ORF4编码产物是TBTV的基因沉默抑制子。Tobacco bushy top virus （TBTV） is a distinct species of the genus Umbravirus, and can cause severe damage to tobacco Plants. The genomic RNA of TBTV contains four putative open reading frames （ORFs）, and the nearly overlapping ORF3 and ORF4 may play important role in the pathoge- nicity of TBTV. The TBTV ORF3 and ORF4 were cloned into the vectors of pGR107 to construct the recombinant express vector of pGR107-ORF3 and pGR107-ORF4, respectively, pGR107-ORF3 or pGRIO7-ORF4 was co-inoculated with pGREEN20$ into the GFP-transformed Nicotiana benthamiana plants. Obvious green fluorescence patterns were observed in the infiltrated areas under long wave ul- traviolet light at 2 to 5 days post-inoculation （dpi）. The green fluorescence areas extended to non-infil- trated zone at 12 dpi on the pGR107-ORF4 inoculated leaves, while the infiltrated zone recovered red and no more green was observed on the pGR107-ORF3 inoculated plants. The results suggested that TBTV ORF4 encoded protein may partially suppress the RNA silencing induced by the GFP-trans- formed N. benthamiana line 16c to the foreign GFP gene, whereas TBTV ORF3 encoded product may not a gene silencing suppressor. Real-time quantitative PCR analysis results revealed that the GFP gene transcripts of the plants infiltrated with pGR107-ORF4 at 15 dpi were higher than that of pGR107 inoc- ulated control, and less lower than that of non-inoculated line 16c. The results further confirmed that TBTV ORF4 product suppressed the RNA silencing triggered by the GFP transgene, and the foreign GFP gene could express normally in the 16c. The TBTV ORF4 encoded protein is a gene silencing suppressor was thus proposed in this study.

关 键 词：烟草丛顶病毒 ORF3 ORF4 沉默抑制子 鉴定 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]