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Jefferson等(1986)提出用β—葡糖苷酸酶(GUS)基因作为基因整合的标志基因。本文用组织化学和荧光定性等方法分析,检验大豆及基野生种(Soja)种子和植株内的类GUS活性。组织化学分析结果表明,栽培大豆的成熟植株和幼苗叶片以及野生种的幼苗叶片均表现为阴性反应。而不同发育时期未成熟豆荚的4个部分(荚皮、种皮、胚乳、胚)都表现强的组织化学着色反应。栽培品种和Soja种的成熟种子的子叶、胚芽、下胚轴、胚根也表现阳性反应。维管束较其周围组织表现出更强的着色反应。大豆类GUS活性存在于成熟干种子的任何部分。着色的深度在种子萌发初期增加,到4天后则迅速下降,到第10天,幼苗的任何部分不再呈现阳性反应。未成熟胚表现强的类GUS着色反应,但在培养1～2天后着色反应消失。定性荧光分析结果表明,各发育时期的大豆种子均表现阴性反应但反应强度不及阳性对照,而阴性对照无反应。用标准的ELISA(依丽沙)方法分析结果表明,所有阳性对照的细胞具有抗GUS活性,而所有样本均无抗GUS活性。这表明大豆种子及其组织内的控制类GUS活性的蛋白质与大肠杆菌的GUS酶没有抗原相似性。因此,在转基因大豆植株里分析大肠杆菌的GUS活性反应是可能的。
Jefferson et al. (1986) proposed the use of the beta-glucuronidase (GUS) gene as a marker for gene integration. In this study, we examined the GUS-like activity in Soja seeds and plants using histochemical and fluorescent qualitative methods. Histochemical analysis showed that the mature plants and seedlings of cultivated soybean leaves and wild seedling leaves showed negative reaction. The four parts of immature pods at different developmental stages (pod, seed coat, endosperm, embryo) showed strong histochemical staining reaction. Cotyledons, germs, hypocotyls, and radicles of mature cultivars and Soja species also showed positive responses. Vascular bundles showed a stronger coloring response than their surrounding tissues. Soybean GUS activity is present in any part of the mature dry seed. The depth of coloration increased at the early germination stage and decreased rapidly after 4 days. By the 10th day, the seedlings no longer showed any positive reaction. Immature embryos showed a strong GUS-like color reaction, but the color reaction disappeared after 1 to 2 days of culture. The results of qualitative fluorescence analysis showed that the soybean seeds in each developmental stage showed negative reaction but the reaction intensity was lower than the positive control, while the negative control did not respond. Analysis using standard ELISA (Elisa) methods showed that all positive control cells had anti-GUS activity, whereas all samples had no anti-GUS activity. This indicates that the protein that controls GUS activity in soybean seeds and their tissues has no antigenic similarity to the E. coli GUS enzyme. Therefore, it is possible to analyze the GUS activity of E. coli in transgenic soybean plants.