大麦黄矮病毒运动蛋白是RNA沉默抑制因子

大麦黄矮病毒(barley yellow dwarf virus,BYDV)属黄症病毒科家族,其基因组包含6个开放阅读框(open reading frames,ORFs).将BYDV的6个基因分别克隆到pWEIMING101载体上,得到重组基因.电击转化农杆菌后,利用农杆菌瞬时表达方法渗透注射转GFP基因的本氏烟草16c植株的叶片,在长波长紫外灯下观察GFP的表达,并通过Northern blot证明所得现象.研究结果表明,BYDV的PAV株系ORF4编码的运动蛋白(movement protein,MP)是RNA沉默抑制因子,其表达可以抑制局部和系统RNA沉默.BYDV-MP与GFP的双链RNA(dsGFP)共表达后仍能抑制RNA沉默,荧光强度与叶片中GFP的mRNA和其沉默降解形成的siRNA的量有对应关系,其N端核定位序列对抑制局部基因沉默起主要作用,第5、6位氨基酸是抑制基因沉默的关键氨基酸.BYDV-MP单独渗透注射的部位均产生细胞死亡.

The Movement Protein of Barley Yellow Dwarf Virus is a Suppressor of RNA Silencing

The genome of barley yellow dwarf virus （BYDV）, a member of the Luteoviddae family, contains 6 open reading frames （ORFs）. We cloned the 60RFs into plasmid of pWEIMING101 and electroporated them into Agrobacte- rium tumefaciens for transient coexpression of BYDV ORFs and GFP in Nicotiana benthamiana line 16c, a transgenic GFP plant. Northern blot analysis revealed the movement protein （MP） encoded by BYDV-PAV ORF4 as a suppressor of both local and systemic GFP RNA silencing and RNA silencing triggered by double-stranded GFP RNA. The fluorescent intensity was associated with steady state levels of GFP mRNAs and its degradation product of siRNAs. The nuclear localization signal in the N terminus of the MP and amino acid 5 and 6 were the functional domain and motif, respectively, essential for inhibiting RNA silencing. Transient expression of BYDV-MP alone in infiltrated tissue resulted in cell death.