Dry I, Krake L, Mullineaux P, Rezaian A
CSIRO Plant Industry, Horticulture Unit, Glen Osmond SA, Australia.
Mol Plant Microbe Interact. 2000 May;13(5):529-37. doi: 10.1094/MPMI.2000.13.5.529.
The regulation of expression of the two virion-sense (V1 and V2) and four complementary-sense (C1, C2, C3, and C4) open reading frames (ORFs) of Tomato leaf curl virus (TLCV) was studied in both stably and transiently transformed Nicotiana tabacum tissues with fusions with the beta-glucuronidase (GUS) reporter gene. GUS-expressing transgenic lines were obtained with each of the four complementary-sense gene-GUS fusion constructs and with truncated versions of the virion-sense gene-GUS fusion constructs (V1GUSdeltaC and V2GUSdeltaC) lacking complementary-sense sequences encoding the C1, C2, and C3 ORFs. However, little or no GUS expression was observed in kanamycin-resistant plants transformed with full-length, virion-sense gene constructs (V1GUS and V2GUS) constituting the complete viral genome. In contrast, V1GUS and V2GUS were found to direct high-level GUS expression in transient assays with tobacco protoplasts, suggesting that integration of viral constructs containing functional, complementary-sense genes may lead to repression or deletion of the introduced constructs in transgenic tissues. V2GUS expression in the transient protoplast assay was found to be severely curtailed by specific mutation of the C2 ORF, supporting a role for the C2 protein in transactivation of TLCV virion-sense gene expression. TLCV ORF-GUS constructs displayed distinctive tissue expression patterns in transgenic tobacco plants that could be divided into constitutive (C1, C4, and V2GUSdeltaC), predominantly vascular (C2, C3), or reduced expression in cells associated with the vascular bundles (V1GUSdeltaC). The significance of these results is discussed in terms of current models of gene function and regulation in geminiviruses.
利用与β-葡萄糖醛酸酶（GUS）报告基因的融合，在稳定和瞬时转化的烟草组织中研究了番茄卷叶病毒（TLCV）的两个病毒链（V1和V2）和四个互补链（C1、C2、C3和C4）开放阅读框（ORF）的表达调控。用四种互补链基因-GUS融合构建体中的每一种以及缺少编码C1、C2和C3 ORF的互补链序列的病毒链基因-GUS融合构建体的截短版本（V1GUSdeltaC和V2GUSdeltaC）获得了表达GUS的转基因株系。然而，在用构成完整病毒基因组的全长病毒链基因构建体（V1GUS和V2GUS）转化的卡那霉素抗性植物中，几乎没有观察到GUS表达。相反，在烟草原生质体的瞬时分析中发现V1GUS和V2GUS可指导高水平的GUS表达，这表明含有功能性互补链基因的病毒构建体整合可能导致转基因组织中导入构建体的抑制或缺失。在瞬时原生质体分析中发现，C2 ORF的特异性突变严重抑制了V2GUS的表达，这支持了C2蛋白在TLCV病毒链基因表达反式激活中的作用。TLCV ORF-GUS构建体在转基因烟草植物中表现出独特的组织表达模式，可分为组成型（C1、C4和V2GUSdeltaC）、主要在维管束中表达（C2、C3）或在与维管束相关的细胞中表达降低（V1GUSdeltaC）。根据双生病毒目前的基因功能和调控模型对这些结果的意义进行了讨论。
