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首页> 外文期刊>Advancements in Life Sciences >Microscopic detection of chloroplast transgenic plastids using fluorescent probe
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Microscopic detection of chloroplast transgenic plastids using fluorescent probe

机译：使用荧光探针镜检叶绿体转基因质体

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Background: Fluorescent marker genes have modernized many areas of molecular biology,specifically in plant biotechnology and genetic engineering studies. The use of fluorescent proteinspermits the scientists to purify the desired clones visually in transformation work. Greenfluorescent protein (gfp) derived from Aequorea victoria has been the most common and favoritefluorescent marker which is being widely used as a visual selection marker gene. It can be easily visualizedunder UV light without the involvement of any substrate and is non-destructive as well.Method: A species-specific chloroplast transformation vector was constructed with gfp as a fluorescentmarker gene. The recombinant vector was biolistically integrated in tobacco plastome and transgenic cellswere initially screened on spectinomycin containing regeneration medium.Results: The successful plastome integration was verified by using cellular DNA from drug resistant clonesin PCR and southern blotting. The expression of gfp in transplastomic clones was microscopicallyinvestigated using simple florescent as well as confocal laser scanning microscopes.Conclusion: Regeneration of transgenic plants was significantly helped by visual identification offluorescent at different stages of development, also enabling to identify the homozygous and heterozygoustissues. No toxic effect of the gfp was observed and lack of toxicity as maintained by normal phenotypicperformance of plants.
机译：背景：荧光标记基因已使分子生物学的许多领域现代化，特别是在植物生物技术和基因工程研究领域。荧光蛋白的使用允许科学家在转化工作中目视纯化所需的克隆。维多利亚水母（Aequorea victoria）衍生的绿色荧光蛋白（gfp）是最常见和最受欢迎的荧光标记，已被广泛用作视觉选择标记基因。方法：以gfp为荧光标记基因，构建物种特异性的叶绿体转化载体。将重组载体生物整合到烟草质体中，并初步在含有壮观霉素的再生培养基中筛选转基因细胞。结果：利用耐药克隆中的细胞DNA通过PCR和Southern印迹验证了质体成功整合。使用简单的荧光显微镜和共聚焦激光扫描显微镜对转质体克隆中gfp的表达进行了显微镜研究。没有观察到gfp的毒性作用，并且没有由植物的正常表型表现所维持的毒性。

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