Stable nuclear transformation of the diatom Phaeodactylum tricornutum
Stable nuclear transformation of the diatom Phaeodactylum tricornutum
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DOI：
10.1007/bf02172403
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发表时间：
1996-10-16
期刊：
影响因子：
--
通讯作者：
Grossman, AR
中科院分区：
文献类型：
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作者：
Apt, KE;KrothPancic, PG;Grossman, AR
已经为硅藻三角褐指藻开发了核转化系统，利用微粒轰击将印度链球菌的 sh ble 基因引入细胞中。 sh ble 基因编码一种蛋白质，该蛋白质赋予对抗生素博莱霉素（Zeocin）和腐草霉素（phleomycin）的抗性。使用含有来自三角褐指藻fcp基因的启动子和终止子序列的嵌合基因来驱动sh ble的表达。每10(8)个细胞回收10-100个转化体。转化体能够在至少 500 μg/ml 的 Zeocin 上生长，这是杀死野生型细胞所需量的 10 倍。根据Southern杂交，shble基因以1-3个拷贝/转化体存在。正确处理的转录本的相对水平与 Sh ble 蛋白的丰度（以 0.1-2.0 μg/mg 总蛋白存在）相关。与 fcp 启动子融合的 cat 报告基因也可以通过微粒轰击引入，并且被发现高度表达（平均为 7.1 U/mg 总蛋白）。这项工作表明异源基因可以很容易地在三角褐藻中表达。选择标记和报告基因构建体的开发为剖析基因结构和调控以及将新功能引入硅藻提供了必要的工具。
A nuclear transformation system has been developed for the diatom Phaeodactylum tricornutum using microparticle bombardment to introduce the sh ble gene from Streptoalloteichus hindustanus into cells. The sh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from the P. tricornutum fcp genes were used to drive expression of sh ble. Between 10-100 transformants were recovered/10(8) cells. Transformants were able to grow on at least 500 mu g/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations the sh ble gene was present in 1-3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1-2.0 mu g/mg total protein). The cat reporter gene fused to a fcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed in P. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.