大麦黄矮病毒GAV株系ORF4基因在杆状病毒-昆虫细胞系统中的表达及亚细胞定位

Expression and Subcellular Localization of the ORF4 gene of Barley Yellow Dwarf Virus GAV Strain in Baculovirus-insect Cell Expression System

摘要:

根据已报道的大麦黄矮病毒GAV株系(BYDV-GAV)相关基因序列，利用RT-PCR方法获得ORF4基因。在杆状病毒-昆虫细胞系统中，成功表达了ORF4和GFP（绿色荧光蛋白）的融合蛋白(GFP: ORF4)，Western blot检测到目的蛋白的表达。利用激光共聚焦显微镜观察其在细胞中的积累和亚细胞分布，发现ORF4基因编码的17 kD蛋白（P4）能进入细胞核，并在细胞核膜上聚集。通过对ORF4基因编码的P4蛋白的N端和C端缺失突变结合蛋白质的结构预测分析，鉴定出N端α螺旋结构对于P4蛋白的核膜定位是必需的。这些结果为进一步研究ORF4基因在黄矮病毒GAV系统侵染中的生物学功能奠定了基础。

Abstract:

According to published nucleotide sequences, ORF4 gene of barley yellow dwarf virus GAV (BYDV-GAV) was synthesized by reverse transcription-polymerase chain reaction (RT-PCR).The BYDV-GAV ORF4 gene was expressed in baculovirus -insect cell expression system efficiently, and western bolt analysis confirmed its expression product. Confocal laser scanning microscopy showed that GFP: ORF4 fusion protein was associated with the nuclear envelope of insect cells. By expressing the N- and C-terminal regions of ORF4-encoding product (P4) in insect cells combined with structure prediction, it was found that the N-terminal region of P4 containing four α-helices is required for targeting P4 to the nuclear envelope. These results provide a base for biological function of ORF4 gene during systemic infection of BYDV-GAV in host plants further.