摘要
马铃薯卷叶病毒(Potato leafroll virus,PLRV)P0是由开放阅读框1(ORF1)所编码,利用农杆菌介导的瞬时表达技术渗透注射转绿色荧光蛋白(GFP)基因的16c烟草叶片发现PLRV-P0能够抑制由GFP mRNA引起的基因沉默,结果表明PLRV-P0是马铃薯卷叶病毒编码的一个基因沉默抑制因子。通过序列分析发现PLRV-P0基因序列中含有两个重复的WG基序,我们将PLRV-P0基因序列中第87位和第140位的色氨酸(W)点突变为丙氨酸(A)(命名为P0WA),构建植物表达载体pCAMBIA1300-CE-P0,pCAMBIA1300-CE-P0WA,农杆菌渗透注射本氏(Nicotiana benthamiana)烟草叶片,通过荧光显微镜观察发现PLRV-P0和AGO共同注射后有绿色荧光出现而PLRV-P0WA和AGO共同注射后则没有绿色荧光出现。研究结果初步表明,PLRV-P0能够和AGO蛋白发生相互作用,重复的WG基序是其与AGO蛋白相互作用的关键氨基酸。
P0 of Potato leafroll virus is encoded by the open reading framel （ ORF1 ） . Here we show that PO of Potato leafroll virus （ PLRV ） displays strong silencing suppressor activity in a transient expressin assay based upon its ability to inhibit PTGS of green flurescent protein （ GFP ） mRNA when expressed in agro-infitraten leaves of Nicotiana benthamiana containing a GFP transgene （ 16c ） .We found PLRV-PO gene sequence containing two duplicate WG motif by sequence analysising. The 87 and 140 Tryptophan （ W ） of Potato leafroll virus PO gene were mutated by Alanine （ A ） （ named POWA ） , the plant expression vector pCAMBIA1300-CE-PO, pCAMBIA13OO-CE-POWA was constructed. We found that there was green fluorescent under fluorescence microscopy after PLRV-PO and AGO co-injection when expressed in agro-infihraten leaves of Nicotiana benthamiana. However, there was not green fluorescent under fluorescence microscopy after PLRV-POWA and AGO co-injection.We conclude that the PLRV-PO interacted with AGO protein and repeated WG motif was the key amino acids.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第3期70-76,共7页
Biotechnology Bulletin
基金
国家自然科学基金项目(30870109)
