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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 readingframes, 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。
Barley yellow dwarf virus (BYDV) belongs to the family of Xylanosis and its genome contains 6 open reading frames (ORFs). Six genes of BYDV were cloned into pWEIMING101 vector respectively to obtain the recombinant gene. After Agrobacterium tumefaciens transformation, Agrobacterium tumefaciens transient expression method was used to infiltrate the leaves of N. benthamiana 16c plants transformed with GFP gene. The expression of GFP was observed under long wavelength ultraviolet light and the result was confirmed by Northern blot. The results showed that movement protein (MP) encoded by ORF4 of PAV strain of BYDV is an inhibitor of RNA silencing, and its expression can inhibit local and systemic RNA silencing. After co-expression of BYDV-MP and GFP double-stranded RNA (dsGFP), RNA silencing was still inhibited. The fluorescence intensity correlated with the amount of GFP mRNA in leaves and the amount of siRNA formed by silencing degradation. The N-terminal nuclear localization sequence Local gene silencing plays a major role. Amino acids at positions 5 and 6 are the key amino acids that suppress gene silencing. BYDV-MP alone infiltrated the site of cell death.