Ascenzi R, Ingram J L, Massel M, Thompson W F, Spiker S, Weissinger A K
Department of Botany, North Carolina State University, Raleigh 27695-7612, USA.
Transgenic Res. 2001 Oct;10(5):465-70. doi: 10.1023/a:1012082602587.
The tobacco nuclear matrix attachment region (MAR), RB7, has been shown to have a much greater effect on transgene expression in cultured cells than in transgenic plants. This is comparable to work in mouse systems showing that MARs have a positive effect on transgene expression in embryonic tissues but not adult tissues. There are several possible explanations for these observations. One is that cell differentiation state and proliferation rate can affect MAR function. We tested this possibility by initiating suspension cell cultures from well-characterized transgenic plants transformed with 35S::GUS with and without flanking MARs and then comparing GUS specific activity in the cell lines to those of the transgenic plants from which the cell lines were derived. If cell differentiation state and proliferation rate do affect MAR function, we would expect the ratio of transgene expression (cell suspensions : plants) to be greater in MAR lines than in control lines. This turned out not to be the case. Thus, it appears that MAR function is not enhanced simply because cells in culture divide rapidly and are not differentiated. Because in animal systems the chromosomal protein HMG-I/Y has been shown to be upregulated in proliferating cells and may have a role in MAR function, we have also examined the levels of the tobacco HMG-I/Y homolog by immunoblotting. The level of this protein does not differ between primary transformant cultured cells (NT-1) and Nicotiana tabacum plants (SR-1). However, a higher molecular weight cross-reacting polypeptide was found in nuclei from the NT-1 cell suspensions but was not detected in SR-1 leaf nuclei or cell suspensions derived from the SR-1 plants.
烟草核基质附着区（MAR）RB7在培养细胞中对转基因表达的影响远大于在转基因植物中的影响。这与小鼠系统中的研究结果类似，即MAR对胚胎组织中的转基因表达有积极作用，但对成体组织则不然。对于这些观察结果有几种可能的解释。一种是细胞分化状态和增殖速率会影响MAR功能。我们通过从用含或不含侧翼MAR的35S::GUS转化的特征明确的转基因植物中启动悬浮细胞培养，然后将细胞系中的GUS比活性与细胞系所源自的转基因植物的GUS比活性进行比较，来测试这种可能性。如果细胞分化状态和增殖速率确实影响MAR功能，我们预期MAR系中转基因表达的比例（细胞悬浮液∶植物）会高于对照系。但结果并非如此。因此，似乎MAR功能不会仅仅因为培养中的细胞快速分裂且未分化就得到增强。因为在动物系统中，已证明染色体蛋白HMG-I/Y在增殖细胞中上调，并且可能在MAR功能中起作用，所以我们也通过免疫印迹检测了烟草HMG-I/Y同源物的水平。这种蛋白在初级转化体培养细胞（NT-1）和烟草植株（SR-1）之间的水平没有差异。然而，在NT-1细胞悬浮液的细胞核中发现了一种分子量更高的交叉反应多肽，但在SR-1叶细胞核或源自SR-1植株的细胞悬浮液中未检测到。
