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小球藻己糖/H⁺同向转运体在团藻中表达时是一种有用的选择标记和生化试剂。

The Chlorella hexose/H+ symporter is a useful selectable marker and biochemical reagent when expressed in Volvox.

作者信息

Hallmann A, Sumper M

机构信息

Lehrstuhl Biochemic I, Universität Regensburg, Germany.

出版信息

Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):669-73. doi: 10.1073/pnas.93.2.669.

Abstract

The multicellular obligately photoautotrophic alga Volvox is composed of only two types of cells, somatic and reproductive. Therefore, Volvox provides the simplest model system for the study of multicellularity. Metabolic labeling experiments using radioactive precursors are crucial for the detection of stage- and cell-type-specific proteins, glycoproteins, lipids, and carbohydrates. However, wild-type Volvox lacks import systems for sugars or amino acids. To circumvent this problem, the hexose/H+ symporter (HUP1) gene from the unicellular alga Chlorella was placed under the control of the constitutive Volvox beta-tubulin promoter. The corresponding transgenic Volvox strain synthesized the sugar transporter in a functional state and was able to efficiently incorporate 14C from labeled glucose or glucosamine. Sensitivity toward the toxic glucose/mannose analogue 2-deoxy-glucose increased by orders of magnitude in transformants. Thus we report the successful transformation of Volvox with a gene of heterologous origin. The chimeric gene may be selected for in either a positive or a negative manner, because transformants exhibit both prolonged survival in the dark in the presence of glucose and greatly increased sensitivity to the toxic sugar 2-deoxyglucose. The former trait may make the gene useful as a dominant selectable marker for use in transformation studies, whereas the latter trait may make it useful in development of a gene-targeting system.

摘要

多细胞专性光合自养藻类团藻仅由体细胞和生殖细胞两种类型的细胞组成。因此,团藻为多细胞性研究提供了最简单的模型系统。使用放射性前体的代谢标记实验对于检测阶段特异性和细胞类型特异性的蛋白质、糖蛋白、脂质和碳水化合物至关重要。然而,野生型团藻缺乏糖类或氨基酸的导入系统。为了解决这个问题,将单细胞藻类小球藻的己糖/H⁺同向转运体(HUP1)基因置于组成型团藻β-微管蛋白启动子的控制之下。相应的转基因团藻菌株合成了处于功能状态的糖转运体,并且能够有效地从标记的葡萄糖或氨基葡萄糖中掺入¹⁴C。在转化体中,对有毒的葡萄糖/甘露糖类似物2-脱氧葡萄糖的敏感性提高了几个数量级。因此,我们报道了用异源基因成功转化团藻。嵌合基因可以以正向或负向方式进行选择,因为转化体在葡萄糖存在下在黑暗中表现出延长的存活时间,并且对有毒糖2-脱氧葡萄糖的敏感性大大增加。前一个特性可能使该基因成为用于转化研究的显性选择标记有用,而后一个特性可能使其在基因靶向系统的开发中有用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9414/40110/6556c73d9ea9/pnas01506-0136-a.jpg

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